RNA modulating oligonucleotides with improved characteristics for the treatment of neuromuscular disorders

ABSTRACT

The current invention provides an improved oligonucleotide and its use for treating, ameliorating, preventing, delaying and/or treating a human cis-element repeat instability associated genetic neuromuscular or neurodegenerative disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/676,569, filed Aug. 14, 2017, which is a division of U.S. patentapplication Ser. No. 14/522,002, filed Oct. 23, 2014, now U.S. Pat. No.9,745,576, which is a continuation of International Patent ApplicationNo. PCT/NL2013/050306, filed Apr. 23, 2013, which claims priority toU.S. Patent Application Ser. No. 61/636,914, filed Apr. 23, 2012, andEuropean Patent Application No. 12165139.2, filed Apr. 23, 2012, thedisclosures of which are incorporated herein by reference in theirentirety.

Reference to Sequence Listing Submitted Electronically

The content of the electronically submitted sequence listing in ASCIItext file (Name: 617845_VCO9-002PCCONDIVCON_Sequence_Listing.txt; Size:298,997 bytes; Created: Oct. 22, 2019) is incorporated herein byreference in its entirety.

FIELD

The invention relates to the field of human genetics, more specificallyneuromuscular disorders. The invention in particular relates to the useof antisense oligonucleotides (AONs) with improved characteristicsenhancing clinical applicability as further defined herein.

BACKGROUND OF THE INVENTION

Neuromuscular diseases are characterized by impaired functioning of themuscles due to either muscle or nerve pathology (myopathies andneuropathies). The neuropathies are characterized by neurodegenerationand impaired nerve control leading to problems with movement, spasticityor paralysis. Examples include Huntington's disease (HD), several typesof spinocerebellar ataxia (SCA), Friedreich's ataxia (FA), AmyotrophicLateral Sclerosis (ALS) and Frontotemporal dementia (FTD). A subset ofneuropathies is caused by a cis-element repeat instability. Forinstance, HD is caused by a triplet (CAG)n repeat expansion in exon 1 ofthe HTT gene. Expansion of these repeats results in expansion of aglutamine stretch at the N-terminal end of the 348 kDa cytoplasmichuntingtin protein. Huntingtin has a characteristic sequence of 6 to 29glutamine amino acid residues in the normal form; the mutated huntingtincausing the disease has more than 38 residues. The continuous expressionof mutant huntingtin molecules in neuronal cells results in theformation of large protein deposits which eventually give rise to celldeath, especially in the frontal lobes and the basal ganglia (mainly inthe caudate nucleus). The severity of the disease is generallyproportional to the number of extra residues. AONs specificallytargeting the expanded CAG repeats (such as PS57 (CUG)₇ as a 2′-O-methylphosphorothioate RNA; SEQ ID NO:1 Evers et al.) can be applied toeffectively reduce mutant huntingtin transcript and (toxic) proteinlevels in HD patient-derived cells. For treatment of neuropathies,systemically administered AONs need to pass the blood brain barrier.Thus, there is a need for optimization of oligochemistry allowing and/orexhibiting improved brain delivery.

The myopathies include genetic muscular dystrophies that arecharacterized by progressive weakness and degeneration of skeletal,heart and/or smooth muscle. Examples of myopathies are Duchenne musculardystrophy (DMD), myotonic dystrophy type 1 (DM1), and myotonic dystrophytype 2 (DM2). DM1 and DM2 are both also caused by cis-element repeatinstability; DM1 by a trinucleotide (CTG)_(n) repeat expansion in the 3′untranslated region of exon 15 in the DMPK gene, and DM2 by atetranucleotide (CCTG)_(n) repeat expansion in the DM2/ZNF9 gene. Alsohere, AONs specifically targeting the expanded repeats, such as PS58,(CAG)₇, a 2′-O-methyl phosphorothioate RNA for DM1 (Mulders et al.),have been shown to efficiently induce the specific degradation of the(toxic) expanded repeat transcripts. In contrast to DMD where the genedefect is associated with increased permeability of the muscle fibermembranes for small compounds as AONs, for most other myopathies anenhanced AON distribution to and uptake by muscle tissue is essential toobtain a therapeutic effect. Thus, also here there is a need foroptimization of oligochemistry allowing and/or exhibiting improvedmuscle delivery.

The particular characteristics of a chosen chemistry at least in partaffect the delivery of an AON to the target transcript: administrationroute, biostability, biodistribution, intra-tissue distribution, andcellular uptake and trafficking. In addition, further optimization ofoligonucleotide chemistry is conceived to enhance binding affinity andstability, enhance activity, improve safety, and/or to reduce cost ofgoods by reducing length or improving synthesis and/or purificationprocedures. Multiple chemical modifications have become generally and/orcommercially available to the research community (such as 2′-O-methylRNA and 5-substituted pyrimidines and 2,6-diaminopurines), whereas mostothers still present significant synthetic effort to obtain. Especiallypreliminary encouraging results have been obtained using 2′-O-methylphosphorothioate RNA containing modifications on the pyrimidine andpurine bases as identified herein.

In conclusion, to enhance the therapeutic applicability of AONs fortreating human cis-element repeat instability associated geneticdisorders as exemplified herein, there is a need for AONs with furtherimproved characteristics.

DESCRIPTION OF THE INVENTION

Oligonucleotide

In a first aspect, the invention provides an oligonucleotide comprising2′-O-methyl RNA nucleotide residues, having a backbone wherein at leastone phosphate moiety is replaced by a phosphorothioate moiety, andcomprising one or more 5-methylpyrimidine and/or one or more2,6-diaminopurine bases; or an oligonucleotide consisting of 2′-O-methylRNA nucleotide residues and having a backbone wherein all phosphatemoieties are replaced by phosphorothioate moieties, and comprising oneor more 5-methylpyrimidine and/or one or more 2,6-diaminopurine bases,for use as a medicament for treating human cis-element repeatinstability associated genetic disorders.

In the context of the invention, “backbone” is used to identify thechain of alternating ribose rings and internucleoside linkages, to whichthe nucleobases are attached. The term “linkage” is used for theconnection between two ribose units (i.e. “internucleoside linkage”),which is generally a phosphate moiety. Thus, an oligonucleotide having10 nucleotides may contain 9 linkages, linking the 10 ribose unitstogether. Additionally, there may be one or more last linkage(s) presentat one or both sides of the oligonucleotide, which is only connected toone nucleotide. The terms “linkage” and “internucleoside linkage” arealso meant to indicate such a pendant linkage. At least one of thelinkages in the backbone of the oligonucleotide according to theinvention consists of a phosphorothioate moiety, linking two riboseunits. Thus, at least one of the naturally occurring 3′ to 5′phosphodiester moieties present in RNA is replaced by a phosphorothioatemoiety.

Within the context of the invention, “a” in each of the followingexpressions means “at least one”: a 2′-O-methyl RNA nucleotide residue,a 2′-O-methyl RNA residue, a phosphorothioate moiety, a 2′-O-methylphosphorothioate RNA residue, a 5-methylpyrimidine base, a5-methylcytosine base, a 5-methyluracil base, a thymine base, a2,6-diaminopurine base.

Preferably, the oligonucleotide according to the invention is anoligonucleotide with less than 37 nucleotides. Said oligonucleotide mayhave 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, or 36 nucleotides. Such oligonucleotide mayalso be identified as an oligonucleotide having from 12 to 36nucleotides.

Accordingly, an oligonucleotide of the invention, comprising a2′-O-methyl RNA nucleotide residue having a backbone wherein at leastone phosphate moiety is replaced by a phosphorothioate moiety, comprisesless than 37 nucleotides (i.e. it comprises 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides) and a 5-methylpyrimidine and/or a 2,6-diaminopurine base.

Accordingly, an oligonucleotide of the invention, consisting of2′-O-methyl RNA nucleotide residues and having a backbone wherein allphosphate moieties are replaced by phosphorothioate, and comprises lessthan 34 nucleotides (i.e. it comprises 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides) and a 5-methylpyrimidine and/or a 2,6-diaminopurine base.

In a preferred embodiment, the oligonucleotide of the inventioncomprises a 2′-(O)-methyl phosphorothioate RNA nucleotide residue, orconsists of 2′-O-methyl phosphorothioate RNA nucleotide residues. Sucholigonucleotide comprises a 2′-(O)-methyl RNA residue, which isconnected through a phosphorothioate linkage to the next nucleotide inthe sequence. This next nucleotide may be, but not necessarily, another2′-O-methyl phosphorothioate RNA nucleotide residue. Alternatively, sucholigonucleotide consists of 2′-O-methyl phosphorothioate RNA nucleotideresidues, wherein all nucleotides comprise a 2′-O-methyl moiety and aphosphorothioate moiety. Preferably, such oligonucleotide consists of2′-O-methyl phosphorothioate RNA nucleotide residues. Such chemistry isknown to the skilled person. Throughout the application, anoligonucleotide comprising a 2′-O-methyl RNA residue and aphosphorothioate linkage may be replaced by an oligonucleotidecomprising a 2′-(O)-methyl phosphorothioate RNA nucleotide residue or anoligonucleotide comprising a 2′-O-methyl phosphorothioate RNA residue.Throughout the application, an oligonucleotide consisting of 2′-O-methylRNA residues linked by or connected through phosphorothioate linkages oran oligonucleotide consisting of 2′-O-methyl phosphorothioate RNAnucleotide residues may be replaced by an oligonucleotide consisting of2′-O-methyl phosphorothioate RNA.

In addition, an oligonucleotide of the invention comprises at least onebase modification that increases binding affinity to target strands,increases melting temperature of the resulting duplex of saidoligonucleotide with its target, and/or decreases immunostimulatoryeffects, and/or increases biostability, and/or improves biodistributionand/or intra-tissue distribution, and/or cellular uptake andtrafficking. In an embodiment, an oligonucleotide of the inventioncomprises a 5-methylpyrimidine and/or a 2,6-diaminopurine base. A5-methylpyrimidine base is selected from a 5-methylcytosine and/or a5-methyluracil and/or a thymine, in which thymine is identical to5-methyluracil. Where an oligonucleotide of the invention has two ormore such base modifications, said base modifications may be identical,for example all such modified bases in the oligonucleotide are5-methylcytosine, or said base modifications may be combinations ofdifferent base modifications, for example the oligonucleotide may haveone or more 5-methylcytosines and one or more 5-methyluracils.

In a preferred embodiment, an oligonucleotide of the invention (i.e. anoligonucleotide comprising 2′-O-methyl RNA nucleotide residues, having abackbone wherein at least one phosphate moiety is replaced by aphosphorothioate moiety, and comprising one or more 5-methylpyrimidineand/or one or more 2,6-diaminopurine bases; or an oligonucleotideconsisting of 2′-O-methyl RNA nucleotide residues and having a backbonewherein all phosphate moieties are replaced by phosphorothioatemoieties, and comprising one or more 5-methylpyrimidine and/or one ormore 2,6-diaminopurine bases) is such that it does not comprise a2′-deoxy 2′-fluoro nucleotide (i.e. 2′-deoxy 2′-fluoro-adenosine,-guanosine, -uridine and/or -cytidine). Such oligonucleotide comprisinga 2′-fluoro (2′-F) nucleotide has been shown to be able to recruit theinterleukin enhancer-binding factor 2 and 3 (ILF2/3) and is thereby ableto induce exon skipping in the targeted pre-mRNA (Rigo F, et al,WO2011/097614). In the current invention, the oligonucleotide usedpreferably does not recruit such factors and/or the oligonucleotide ofthe invention does not form heteroduplexes with RNA that arespecifically recognized by the ILF2/3. The mechanism of action of theoligonucleotide of the current invention is assumed to be distinct fromthe one of an oligonucleotide with a 2′-F nucleotide: theoligonucleotide of the invention is expected to primarily induce thespecific degradation of the (toxic) expanded repeat transcripts.

‘Thymine’ and ‘5-methyluracil’ may be interchanged throughout thedocument. In analogy, 2,6-diaminopurine is identical to 2-aminoadenineand these terms may be interchanged throughout the document.

The term “base modification” or “modified base” as identified hereinrefers to the modification of a naturally occurring base in RNA (i.e.pyrimidine or purine base) or to the de novo synthesis of a base. Thisde novo synthesized base could be qualified as “modified” by comparisonto an existing base.

An oligonucleotide of the invention comprising a 5-methylcytosine and/ora 5-methyluracil and/or a 2,6-diaminopurine base means that at least oneof the cytosine nucleobases of said oligonucleotide has been modified bysubstitution of the proton at the 5-position of the pyrimidine ring witha methyl group (i.e. a 5-methylcytosine), and/or that at least one ofthe uracil nucleobases of said oligonucleotide has been modified bysubstitution of the proton at the 5-position of the pyrimidine ring witha methyl group (i.e. a 5-methyluracil), and/or that at least one of theadenine nucleobases of said oligonucleotide has been modified bysubstitution of the proton at the 2-position with an amino group (i.e. a2,6-diaminopurine), respectively. Within the context of the invention,the expression “the substitution of a proton with a methyl group inposition 5 of the pyrimidine ring” may be replaced by the expression“the substitution of a pyrimidine with a 5-methylpyrimidine,” withpyrimidine referring to only uracil, only cytosine or both. Likewise,within the context of the invention, the expression “the substitution ofa proton with an amino group in position 2 of adenine” may be replacedby the expression “the substitution of an adenine with a2,6-diaminopurine.” If said oligonucleotide comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or more cytosines, uracils, and/or adenines, at least 1, 2, 3,4, 5, 6, 7, 8, 9 or more cytosines, uracils and/or adenines respectivelyhave been modified this way. Preferably all cytosines, uracils and/oradenines have been modified this way or replaced by 5-methylcytosine,5-methyluracil and/or 2,6-diaminopurine, respectively. No need to saythat the invention could only be applied to oligonucleotides comprisingat least one cytosine, uracil, or adenine, respectively, in theirsequence.

We discovered that the presence of a 5-methylcytosine, 5-methyluraciland/or a 2,6-diaminopurine in an oligonucleotide of the invention has apositive effect on at least one of the parameters or an improvement ofat least one parameters of said oligonucleotides. In this context,parameters may include: binding affinity and/or kinetics, silencingactivity, biostability, (intra-tissue) distribution, cellular uptakeand/or trafficking, and/or immunogenicity of said oligonucleotide, asexplained below.

Binding affinity and kinetics depend on the AON's thermodynamicproperties. These are at least in part determined by the meltingtemperature of said oligonucleotide (Tm; calculated with e.g. theoligonucleotide properties calculator(www.unc.edu/∫cail/biotool/oligo/index.html oreu.idtdna.com/analyzer/Applications/OligoAnalyzer/) for single strandedRNA using the basic Tm and the nearest neighbor model), and/or the freeenergy of the oligonucleotide-target exon complex (using RNA structureversion 4.5 or RNA mfold version 3.5). If a Tm is increased, the exonskipping activity typically increases, but when a Tm is too high, theAON is expected to become less sequence-specific. An acceptable Tm andfree energy depend on the sequence of the oligonucleotide. Therefore, itis difficult to give preferred ranges for each of these parameters.

An activity of an oligonucleotide of the invention is to inhibit theformation of a mutant protein and/or silence or reduce or decrease thequantity of a disease-associated or disease-causing or mutant transcriptcontaining an extended or unstable number of repeats in a cell of apatient, in a tissue of a patient and/or in a patient as explained laterherein. An oligonucleotide of the invention comprising or consisting ofa 2′-O-methyl phosphorothioate RNA and a 5-methylcytosine and/or a5-methyluracil and/or a 2,6-diaminopurine base is expected to be able tosilence or reduce or decrease the quantity of said transcript moreefficiently than what an oligonucleotide comprising or consisting of a2′-O-methyl phosphorothioate RNA but without any 5-methylcytosine,without any 5-methyluracil and without any 2,6-diaminopurine base willdo. This difference in terms of efficiency may be of at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%. The reduction or decrease may be assessed byNorthern Blotting or (semi-) quantitative RT-PCR for transcript levels(preferably as carried out in the experimental part) or by Westernblotting for protein levels. An oligonucleotide of the invention mayfirst be tested in the cellular system like patient-derived fibroblastsas described in Example 1.

Biodistribution and biostability are preferably at least in partdetermined by a validated hybridization ligation assay adapted from Yuet al., 2002. In an embodiment, plasma or homogenized tissue samples areincubated with a specific capture oligonucleotide probe. Afterseparation, a DIG-labeled oligonucleotide is ligated to the complex anddetection followed using an anti-DIG antibody-linked peroxidase.Non-compartmental pharmacokinetic analysis is performed using WINNONLINsoftware package (model 200, version 5.2, Pharsight, Mountainview,Calif.). Levels of AON (ug) per mL plasma or mg tissue are monitoredover time to assess area under the curve (AUC), peak concentration(C_(max)), time to peak concentration (T_(max)), terminal half life andabsorption lag time (t_(lag)). Such a preferred assay has been disclosedin the experimental part.

AONs may stimulate an innate immune response by activating the Toll-likereceptors (TLR), including TLR9 and TLR7 (Krieg et al., 1995). Theactivation of TLR9 typically occurs due to the presence ofnon-methylated CG sequences present in oligodeoxynucleotides (ODNs), bymimicking bacterial DNA which activates the innate immune system throughTLR9-mediated cytokine release. The 2′-O-methyl modification is howeversuggested to markedly reduce such possible effect. TLR7 has beendescribed to recognize uracil repeats in RNA (Diebold et al., 2006).

Activation of TLR9 and TLR7 result in a set of coordinated immuneresponses that include innate immunity (macrophages, dendritic cells(DC), and NK cells)(Krieg et al., 1995; Krieg, 2000). Several chemo- andcytokines, such as IP-10, TNFα, IL-6, MCP-1 and IFNα (Wagner, 1999;Popovic et al., 2006) have been implicated in this process. Theinflammatory cytokines attract additional defensive cells from theblood, such as T and B cells. The levels of these cytokines can beinvestigated by in vitro testing. In short, human whole blood isincubated with increasing concentrations of AONs after which the levelsof the cytokines are determined by standard commercially available ELISAkits. A decrease in immunogenicity preferably corresponds to adetectable decrease of concentration of at least one of the cytokinesmentioned above by comparison to the concentration of correspondingcytokine in an assay in a cell treated with an oligonucleotidecomprising at least one 5-methylcytosine and/or 5-methyluracil, and/or2,6-diaminopurine compared to a cell treated with a correspondingoligonucleotide having no 5-methylcytosines, 5-methyluracils, or2,6-diaminopurines.

Accordingly, a preferred oligonucleotide of the invention has animproved parameter, such as an acceptable or a decreased immunogenicityand/or a better biodistribution and/or acceptable or improved RNAbinding kinetics and/or thermodynamic properties by comparison to acorresponding oligonucleotide consisting of a 2′-O-methylphosphorothioate RNA without a 5-methylcytosine, without a5-methyluracil and without a 2,6-diaminopurine. Each of these parameterscould be assessed using assays known to the skilled person or preferablyas disclosed herein.

Below other chemistries and modifications of the oligonucleotide of theinvention are defined. These additional chemistries and modificationsmay be present in combination with the chemistry already defined forsaid oligonucleotide, i.e. the presence of a 5-methylcytosine, a5-methyluracil and/or a 2,6-diaminopurine, and the oligonucleotidecomprising or consisting of 2′-O-methyl phosphorothioate RNA.

A preferred oligonucleotide of the invention comprises or consists of anRNA molecule or a modified RNA molecule. In a preferred embodiment, anoligonucleotide is single stranded. The skilled person will understandthat it is however possible that a single stranded oligonucleotide mayform an internal double stranded structure. However, thisoligonucleotide is still named a single stranded oligonucleotide in thecontext of this invention. A single stranded oligonucleotide has severaladvantages compared to a double stranded siRNA oligonucleotide: (i) itssynthesis is expected to be easier than two complementary siRNA strands;(ii) there is a wider range of chemical modifications possible toenhance uptake in cells, a better (physiological) stability and todecrease potential generic adverse effects; (iii) siRNAs have a higherpotential for non-specific effects (including off-target genes) andexaggerated pharmacology (e.g. less control possible of effectivenessand selectivity by treatment schedule or dose) and (iv) siRNAs are lesslikely to act in the nucleus and cannot be directed against introns.

In addition to the modifications described above, the oligonucleotide ofthe invention may comprise further modifications such as different typesof nucleic acid nucleotide residues or nucleotides as described below.Different types of nucleic acid nucleotide residues may be used togenerate an oligonucleotide of the invention. Said oligonucleotide mayhave at least one backbone modification (internucleoside linkage and/orsugar modification) and/or at least one base modification compared to anRNA-based oligonucleotide.

A base modification includes a modified version of the natural purineand pyrimidine bases (e.g. adenine, uracil, guanine, cytosine, andthymine), such as hypoxanthine (e.g. inosine), orotic acid, agmatidine,lysidine, pseudouracil, 2-thiopyrimidine (e.g. 2-thiouracil,2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine(e.g. 5-halouracil, 5-propynyluracil, 5-propynylcytosine,5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine,5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine,7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine,8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine,or derivatives thereof; N²-cyclopentylguanine (cPent-G),N²-cyclopentyl-2-aminopurine (cPent-AP), and N²-propyl-2-aminopurine(Pr-AP), or derivatives thereof; and degenerate or universal bases, like2,6-difluorotoluene or absent bases like abasic sites (e.g.1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; orpyrrolidine derivatives in which the ring oxygen has been replaced withnitrogen (azaribose)). Examples of derivatives of Super A, Super G andSuper T can be found in U.S. Pat. No. 6,683,173 (Epoch Biosciences),which is incorporated here entirely by reference. cPent-G, cPent-AP andPr-AP were shown to reduce immunostimulatory effects when incorporatedin siRNA (Peacock H. et al.).

In an embodiment, an oligonucleotide of the invention comprises anabasic site or an abasic monomer. Within the context of the invention,such monomer may be called an abasic site or an abasic monomer. Anabasic monomer or abasic site is a nucleotide residue or building blockthat lacks a nucleobase by comparison to a corresponding nucleotideresidue comprising a nucleobase. Within the invention, an abasic monomeris thus a building block part of an oligonucleotide but lacking anucleobase. Such abasic monomer may be present or linked or attached orconjugated to a free terminus of an oligonucleotide.

In a more preferred embodiment, an oligonucleotide of the inventioncomprises 1-10 or more abasic monomers. Therefore, 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more abasic monomers may be present in an oligonucleotide ofthe invention.

An abasic monomer may be of any type known and conceivable by theskilled person, non-limiting examples of which are depicted below:

Herein, R₁ and R₂ are independently H, an oligonucleotide or otherabasic site(s), provided that not both R₁ and R₂ are H and R₁ and R₂ arenot both an oligonucleotide. An abasic monomer(s) can be attached toeither or both termini of the oligonucleotide as specified before. Itshould be noted that an oligonucleotide attached to one or two an abasicsite(s) or abasic monomer(s) may comprise less than 12 nucleotides. Inthis respect, the oligonucleotide according to the invention maycomprise at least 12 nucleotides, optionally including one or moreabasic sites or abasic monomers at one or both termini.

In the sequence listing, an oligonucleotide of the invention comprisingan abasic monomer may be represented by its nucleotide or base sequence;the abasic monomer not being represented since it may be considered aslinked or attached or conjugated to a free terminus of anoligonucleotide. This is the case for base sequences SEQ ID NO: 107 and108. In table 2, the full sequence of preferred oligonucleotidescomprising SEQ ID NO:107 or 108 is provided: such oligonucleotidecomprises SEQ ID NO: 107 or 108 and 4 abasic monomers at the 3′ terminusof the corresponding SEQ ID NO: 107 or 108. SEQ ID NO: 220 and 221correspond to SEQ ID NO: 107 and 108 further comprising 4 additionalabasic monomers at the 3′ terminus of the oligonucleotide.

When an abasic monomer is present within a base sequence of anoligonucleotide, said abasic monomer is identified in the sequencelisting as part of the sequence of said oligonucleotide as in SEQ IDNO:210 and 213.

In tables 1 and 2, an abasic monomer is identified using the letter Q.

Depending on its length an oligonucleotide of the invention may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 basemodifications. It is also encompassed by the invention to introduce morethan one distinct base modification in said oligonucleotide.

A “sugar modification” indicates the presence of a modified version ofthe ribosyl moiety as naturally occurring in RNA (i.e. the furanosylmoiety), such as bicyclic sugars, tetrahydropyrans, morpholinos,2′-modified sugars, 4′-modified sugars, 5′-modified sugars, and4′-substituted sugars. Examples of suitable sugar modifications include,but are not limited to, 2′-O-modified RNA nucleotide residues, such as2′-O-alkyl or 2′-O-(substituted)alkyl e.g. 2′-O-methyl,2′-O-(2-cyanoethyl), 2′-O-(2-methoxy)ethyl (2′-MOE),2′-O-(2-thiomethyl)ethyl, 2′-O-butyryl, 2′-O-propargyl, 2′-O-allyl,2′-O-(2-amino)propyl, 2′-O-(2-(dimethylamino)propyl),2′-O-(2-amino)ethyl, 2′-O-(2-(dimethylamino)ethyl); 2′-deoxy (DNA);2′-O-(haloalkoxy)methyl (Arai K. et al.) e.g.2′-O-(2-chloroethoxy)methyl (MCEM), 2′-O-(2,2-dichloroethoxy)methyl(DCEM); 2′-O-alkoxycarbonyl e.g. 2′-O-[2-(methoxycarbonyl)ethyl] (MOCE),2′-O-[2-(N-methylcarbamoyl)ethyl] (MCE),2′-O-[2-(N,N-dimethylcarbamoyl)ethyl](DCME); 2′-halo e.g. 2′-F, FANA(2′-F arabinosyl nucleic acid); carbasugar and azasugar modifications;3′-O-alkyl e.g. 3′-O-methyl, 3′-O-butyryl, 3′-O-propargyl, 5′-alkyl e.g.5′-methyl; and their derivatives.

Another sugar modification includes “bridged” or “bicylic” nucleic acid(BNA), e.g. locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt(2′-0,4′-C constrained ethyl) LNA, cMOEt (2′-0,4′-C constrainedmethoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), BNA^(NC)[N-Me](as described in Chem. Commun. 2007, 3765, which is incorporated in itsentirety by reference); tricyclo DNA (tcDNA); unlocked nucleic acid(UNA); 5′-methyl substituted BNAs (as described in U.S. patentapplication Ser. No. 13/530,218, which is incorporated in its entiretyby reference); cyclohexenyl nucleic acid (CeNA), altriol nucleic acid(ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA),pyranosyl-RNA (p-RNA), 3′-deoxypyranosyl-DNA (p-DNA); morpholino (ase.g. in PMO, PMOPlus, PMO-X) and their derivatives, preferably lockednucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2′-0,4′-Cconstrained ethyl) LNA, cMOEt (2′-0,4′-C constrained methoxyethyl) LNA,ethylene-bridged nucleic acid (ENA), tricyclo DNA (tcDNA); cyclohexenylnucleic acid (CeNA), altriol nucleic acid (ANA), hexitol nucleic acid(HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA),3′-deoxypyranosyl-DNA (p-DNA); morpholino (as e.g. in PMO, PMOPlus,PMO-X) and their derivatives. A preferred tcDNA is tc-PS-DNA (tricycloDNA comprising phosphorothioate intemucleoside linkage). Depending onits length, an oligonucleotide of the invention may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 sugarmodifications. It is also encompassed by the invention to introduce morethan one distinct sugar modification in said oligonucleotide. In anembodiment, an oligonucleotide as defined herein comprises or consistsof an LNA or a derivative thereof. BNA derivatives are for exampledescribed in WO 2011/097641, which is incorporated in its entirety byreference. In a more preferred embodiment, an oligonucleotide of theinvention is fully 2′-O-methyl modified. Examples of PMO-X are describedin WO2011150408, which is incorporated here in its entirety byreference.

In a preferred embodiment, the oligonucleotide according to theinvention comprises, apart from the mandatory 2′-O-methyl sugarmodification, at least one other sugar modification selected from2′-O-methyl, 2′-O-(2-methoxy)ethyl, morpholino, a bridged nucleotide orBNA, or the oligonucleotide comprises both bridged nucleotides and2′-deoxy modified nucleotides (BNA/DNA mixmers). More preferably, theoligonucleotide according to the invention is modified over its fulllength with a sugar modification selected from 2′-O-methyl,2′-O-(2-methoxy)ethyl, morpholino, bridged nucleic acid (BNA) or BNA/DNAmixmer.

In a more preferred embodiment, the oligonucleotide according to theinvention comprises is fully 2′-O-methyl modified, preferably fully2′-O-methyl phosphorothioate modified.

A “backbone modification” indicates the presence of a modified versionof the ribosyl moiety (“sugar modification”), as indicated above, and/orthe presence of a modified version of the phosphodiester as naturallyoccurring in RNA (“intemucleoside linkage modification”). Examples ofintemucleoside linkage modifications, which are compatible with thepresent invention, are phosphorothioate (PS), chirally purephosphorothioate, phosphorodithioate (PS2), phosphonoacetate (PACE),phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide,phosphorothioate prodrug, H-phosphonate, methyl phosphonate, methylphosphonothioate, methyl phosphate, methyl phosphorothioate, ethylphosphate, ethyl phosphorothioate, boranophosphate,boranophosphorothioate, methyl boranophosphate, methylboranophosphorothioate, methyl boranophosphonate, methylboranophosphonothioate, and their derivatives. Another modificationincludes phosphoramidite, phosphoramidate, N3′→P5′ phosphoramidate,phosphordiamidate, phosphorothiodiamidate, sulfamate,dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate,methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and theirderivatives. Depending on its length, an oligonucleotide of theinvention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, or 35 backbone modifications. It is also encompassed by theinvention to introduce more than one distinct backbone modification insaid oligonucleotide.

An oligonucleotide of the invention comprises at least onephosphorothioate modification. In a more preferred embodiment, anoligonucleotide of the invention is fully phosphorothioate modified.

Other chemical modifications of an oligonucleotide of the inventioninclude peptide-base nucleic acid (PNA), boron-cluster modified PNA,pyrrolidine-based oxy-peptide nucleic acid (POPNA), glycol- orglycerol-based nucleic acid (GNA), threose-based nucleic acid (TNA),acyclic threoninol-based nucleic acid (aTNA), morpholino-basedoligonucleotide (PMO, PPMO, PMO-X), cationic morpholino-based oligomers(PMOPlus), oligonucleotides with integrated bases and backbones (ONIBs),pyrrolidine-amide oligonucleotides (POMs); and their derivatives.

In another embodiment, an oligonucleotide comprises a peptide nucleicacid and/or a morpholino phosphorodiamidate or a derivative thereof.

Thus, the preferred oligonucleotide according to one aspect of theinvention comprises:

-   -   (a) at least one base modification selected from        5-methylpyrimidine and 2,6-diaminopurine; and/or    -   (b) at least one sugar modification, which is 2′-O-methyl,        and/or    -   (c) at least one backbone modification, which is        phosphorothioate.

Thus, a preferred oligonucleotide according to this aspect of theinvention comprises a base modification (a) and no sugar modification(b) and no backbone modification (c). Another preferred oligonucleotideaccording to this aspect of the invention comprises a sugar modification(b) and no base modification (a) and no backbone modification (c).Another preferred oligonucleotide according to this aspect of theinvention comprises a backbone modification (c) and no base modification(a) and no sugar modification (b). Also oligonucleotides having none ofthe above-mentioned modifications are understood to be covered by thepresent invention, as well as oligonucleotides comprising two, i.e. (a)and (b), (a) and (c) and/or (b) and (c), or all three of themodifications (a), (b) and (c), as defined above. In another preferredembodiment, any of the oligonucleotides as described in the previousparagraph may comprise:

-   -   (a) at least one (additional) base modification selected from        2-thiouracil, 2-thiothymine, 5-methylcytosine, 5-methyluracil,        thymine, 2,6-diaminopurine; and/or    -   (b) at least one (additional) sugar modification selected from        2′-O-methyl, 2′-O-(2-methoxy)ethyl, 2′-deoxy (DNA), morpholino,        a bridged nucleotide or BNA, or the oligonucleotide comprises        both bridged nucleotides and 2′-deoxy modified nucleotides        (BNA/DNA mixmers); and/or    -   (c) at least one (additional) backbone modification selected        from (another) phosphorothioate or phosphordiamidate.

In another preferred embodiment, the oligonucleotide according to theinvention is modified over its entire length with one or more of thesame modification, selected from (a) one of the base modifications;and/or (b) one of the sugar modifications; and/or (c) one of thebackbone modifications.

With the advent of nucleic acid mimicking technology, it has becomepossible to generate molecules that have a similar, preferably the samehybridization characteristics in kind not necessarily in amount asnucleic acid itself. Such functional equivalents are of course alsosuitable for use in the invention.

The skilled person will understand that not each sugar, base, and/orbackbone may be modified the same way. Several distinct modified sugars,bases and/or backbones may be combined into one single oligonucleotideof the invention.

A person skilled in the art will also recognize that there are manysynthetic derivatives of oligonucleotides.

Preferably, said oligonucleotide comprises RNA, as RNA/RNA duplexes arevery stable. It is preferred that an RNA oligonucleotide comprises amodification providing the RNA with an additional property, for instanceresistance to endonucleases, exonucleases, and RNaseH, additionalhybridisation strength, increased stability (for instance in a bodilyfluid), increased or decreased flexibility, increased activity, reducedtoxicity, increased intracellular transport, tissue-specificity, etc. Inaddition, the mRNA complexed with the oligonucleotide of the inventionis preferably not susceptible to RNaseH cleavage. Preferredmodifications have been identified above.

Oligonucleotides containing at least in part naturally occurring DNAnucleotides are useful for inducing degradation of DNA-RNA hybridmolecules in the cell by RNase H activity (EC.3.1.26.4).

Naturally occurring RNA ribonucleotides or RNA-like syntheticribonucleotides comprising oligonucleotides are encompassed herein toform double stranded RNA-RNA hybrids that act as enzyme-dependentantisense through the RNA interference or silencing (RNAi/siRNA)pathways, involving target RNA recognition through sense-antisensestrand pairing followed by target RNA degradation by the RNA-inducedsilencing complex (RISC).

Alternatively or in addition, an oligonucleotide can interfere with theprocessing or expression of precursor RNA or messenger RNA (stericblocking, RNaseH independent processes) in particular but not limited toRNA splicing and exon skipping, by binding to a target sequence of RNAtranscript and getting in the way of processes such as translation orblocking of splice donor or splice acceptor sites. Moreover, theoligonucleotide may inhibit the binding of proteins, nuclear factors andothers by steric hindrance and/or interfere with the authentic spatialfolding of the target RNA and/or bind itself to proteins that originallybind to the target RNA and/or have other effects on the target RNA,thereby contributing to the destabilization of the target RNA,preferably pre-mRNA, and/or to the decrease in amount of diseased ortoxic transcript and/or protein in diseases like HD as identified laterherein.

As herein defined, an oligonucleotide may comprise nucleotides with(RNaseH resistant) chemical substitutions at least one of its 5′ or 3′ends, to provide intracellular stability, and comprises less than 9,more preferably less than 6 consecutive (RNaseH-sensitive) deoxyribosenucleotides in the rest of its sequence. The rest of the sequence ispreferably the center of the sequence. Such oligonucleotide is called agapmer.

Gapmers have been extensively described in WO 2007/089611. Gapmers aredesigned to enable the recruitment and/or activation of RNaseH. Withoutwishing to be bound by theory, it is believed that RNaseH is recruitedand/or activated via binding to the central region of the gapmer made ofdeoxyriboses. An oligonucleotide of the invention which is preferablysubstantially independent of or independent of RNaseH is designed inorder to have a central region which is substantially not able or is notable to recruit and/or activate RNaseH. In a preferred embodiment, therest of the sequence of said oligonucleotide, more preferably itscentral part comprises less than 9, 8, 7, 6, 5, 4, 3, 2, 1, or nodeoxyribose. Accordingly, this oligonucleotide of the invention ispreferably partly to fully replaced as earlier defined herein. “Partlyreplaced” means that the oligonucleotide comprises at least some ofnucleotides that have been replaced, preferably at least 50% of itsnucleotides have been replaced, or at least 55%, 60%, 65%, 70%, 75%,80%, 85%, 90% or 95% have been replaced. 100% replacement of nucleotidescorresponds to “fully replaced”.

Accordingly, the invention provides an oligonucleotide comprising a2′-O-methyl phosphorothioate RNA residue or consisting of 2′-O-methylphosphorothioate RNA and comprising a 5-methylpyrimidine and/or a2,6-diaminopurine base. Most preferably, this oligonucleotide consistsof 2′-O-methyl RNA residues connected through a phosphorothioatebackbone and all of its cytosines and/or all of its uracils and/or allof its adenines, independently, have been replaced by 5-methylcytosine,5-methyluracil and/or 2,6-diaminopurine, respectively. Thus, anoligonucleotide of the invention may have:

At least one and preferably all cytosines replaced with5-methylcytosines,

At least one and preferably all cytosines replaced with5-methylcytosines and at least one and preferably all uracils replacedwith 5-methyluracils,

At least one and preferably all cytosines replaced with5-methylcytosines and at least one and preferably all adenines replacedwith 2,6-diaminopurines,

At least one and preferably all cytosines replaced with5-methylcytosines and at least one and preferably all uracils replacedwith 5-methyluracils and at least one and preferably all adeninesreplaced with 2,6-diaminopurines,

At least one and preferably all uracils replaced with 5-methyluracils,

At least one and preferably all uracils replaced with 5-methyluracilsand at least one and preferably all adenines replaced with2,6-diaminopurines, or

At least one and preferably all adenines replaced with2,6-diaminopurines.

An oligonucleotide of the invention is for use as a medicament forpreventing delaying and/or treating a human cis-element repeatinstability associated genetic disorders preferably as exemplifiedherein. A human cis-element repeat instability associated geneticdisorders as identified herein is preferably a neuromuscular disorder.Preferably said oligonucleotide is for use in therapeutic RNAmodulation. Therefore, the oligonucleotide according to the inventionmay be described as an antisense oligonucleotide (AON). An antisenseoligonucleotide is an oligonucleotide which binds (or is able to bind),targets, hybridizes to (or is able to hybridize to) and/or is reversecomplementary to a specific sequence of a transcript of a gene which isknown to be associated with or involved in a human cis-element repeatinstability associated genetic neuromuscular disorder.

According to the invention, an antisense oligonucleotide comprising orconsisting of 2′-O-methyl RNA nucleotide residues, having a backbonewherein at least one phosphate moiety is replaced by a phosphorothioatemoiety, and further comprising at least one of a 5-methylcytosine and/ora 5-methyluracil and/or a 2,6-diaminopurine, is represented by anucleotide sequence comprising or consisting of a sequence that binds(or is able to bind), hybridizes (or is able to hybridize), targetsand/or is reverse complementary to a repetitive element in a RNAtranscript having as repetitive nucleotide unit a repetitive nucleotideunit, which is selected from the (CAG)_(n), (GCG)_(n), (CGG)_(n),(GAA)_(n), (GCC)_(n), (CCG)_(n), (AUUCU)_(n), (GGGGCC)_(n) or(CCUG)_(n). Said oligonucleotide is preferably a single strandedoligonucleotide.

Although it is to be understood that an oligonucleotide of the inventionbinds (or is able to bind), hybridizes (or is able to hybridize),targets and/or is reverse complementary to a repetitive element presentin a RNA transcript as identified above, it can not be ruled out thatsuch oligonucleotide may also interfere with or bind (or is able tobind) or hybridize to (or is able to hybridize) a corresponding DNA,this RNA transcript is derived from.

A repeat or repetitive element or repetitive sequence or repetitivestretch is herein defined as a repetition of at least 3, 4, 5, 10, 100,1000 or more, of a repetitive unit or repetitive nucleotide unit orrepeat nucleotide unit (as (CAG)_(n), (GCG)_(n), (CGG)_(n), (GAA)_(n),(GCC)_(n), (CCG)_(n), (AUUCU)_(n), (GGGGCC)_(n) or (CCUG)_(n)),comprising a trinucleotide repetitive unit, or alternatively a 4, 5 or 6nucleotide repetitive unit, in a transcribed gene sequence in the genomeof a subject, including a human subject. Accordingly, n is an integerand may be at least 3, 4, 5, 10, 100, 1000 or more. The invention is notlimited to exemplified repetitive nucleotide units. Other repetitivenucleotide unit could be found on the following siteneuromuscular.wustl.edu/mother/dnarep.htm. In the majority of patients,a 30 “pure” repeat or repetitive element or repetitive sequence orrepetitive stretch as identified above (as (CAG)_(n), (GCG)_(n),(CGG)_(n), (GAA)_(n), (GCC)_(n), (CCG)_(n), (AUUCU)_(n), (GGGGCC)_(n) or(CCUG)_(n)) is present in a transcribed gene sequence in the genome ofsaid patient. However, it is also encompassed by the invention, that insome patients, said repeat or repetitive element or repetitive sequenceor repetitive stretch as identified above is not qualified as “pure” oris qualified as a “variant” when for example said repeat or repetitiveelement or repetitive sequence or repetitive stretch as identified aboveis interspersed with at least 1, 2, or 3 nucleotide(s) that do not fitthe nucleotide(s) of said repeat or repetitive element or repetitivesequence or repetitive stretch (Braida C., et al,).

An oligonucleotide according to the invention therefore may not need tobe 100% reverse complementary to a targeted repeat. Usually anoligonucleotide of the invention may be at least 90%, 95%, 97%, 99% or100% reverse complementary to a targeted repeat.

In an embodiment, an antisense oligonucleotide comprises or consists of2′-O-methyl phosphorothioate RNA, comprises a 5-methylcytosine and/or a5-methyluracil and/or a 2,6-diaminopurine, is represented by anucleotide sequence comprising or consisting of a sequence that binds(or is able to bind), hybridizes (or is able to hybridize), targetsand/or is reverse complementary to a (CAG)_(n) tract in a transcript andis particularly useful for the treatment, delay, amelioration and/orprevention of the human genetic diseases Huntington's disease (HD),spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 12 or 17, amyotrophiclateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinaland bulbar muscular atrophy (SBMA) and/or dentatorubropallidoluysianatrophy (DRPLA) caused by CAG repeat expansions in the transcripts ofthe HTT (SEQ ID NO: 80), ATXN1 (SEQ ID NO:81), ATXN2 (SEQ ID NO: 82)ATXN3 (SEQ ID NO: 83), CACNA1A (SEQ ID NO:84), ATXN7 (SEQ ID NO: 85),PPP2R2B (SEQ ID NO: 86), TBP (SEQ ID NO: 87), AR (SEQ ID NO: 88) or ATN1(SEQ ID NO: 89) genes.

Preferably, these genes are from human origin. In this embodiment, anoligonucleotide comprises or consists of 2′-O-methyl phosphorothioateRNA, comprises a 5-methylcytosine and/or a 5-methyluracil and/or a2,6-diaminopurine, is represented by a nucleotide sequence comprising orconsisting of a sequence that binds (or is able to bind), hybridizes (oris able to hybridize), targets and/or is reverse complementary to a(CAG)_(n) repeat as identified above and has as repetitive nucleotideunit (CUG)_(m). The m in (CUG)_(m) is preferably an integer which is 4,5, 6, 7, 8, 9, 10, 11, 12. In a preferred embodiment, m is 5 or 6 or 7or 8 or 9 or 10 or 11 or 12.

It is to be noted that for ALS and FTD, it is known that at least twodistinct repeats in at least two distinct transcripts may be involved ormay be responsible or linked with the disease. One has been identifiedin the previous paragraph (i.e. (CAG)_(n) in a ATXN2 transcript).Another one is being identified later as a (GGGGCC)_(n) repeat or tractin a C9ORF72 transcript. It means that for each of these two diseases,one may envisage to use either one of these two distinctoligonucleotides of the invention to specifically induce the specificdegradation of the corresponding (toxic) expanded repeat transcripts.

Throughout the application, an oligonucleotide defined as being reversecomplementary to, binding (being able to bind), hybridizing (being ableto hybridize) or targeting a repeat as identified above and has orcomprises a repetitive nucleotide unit may have any length comprisedfrom 12 to 36 nucleotides. If we take the example of CUG as repetitivenucleotide unit comprised within said oligonucleotide, anyoligonucleotide comprising UGC or GCU as repetitive nucleotide unit isalso encompassed by the present invention. Depending on the length ofsaid oligonucleotide (for example from 12 to 36 nucleotides), the givenrepetitive nucleotide unit may not be complete at the 5′ and/or at the3′ side of said oligonucleotide. Each of said oligonucleotide isencompassed within the scope of said invention.

Alternatively, if we still take as an example the oligonucleotide havingCUG as repetitive nucleotide unit, it may be represented byH-(P)_(p)-(CUG)_(m)-(Q)_(q)-H, wherein m is an integer as defined above.Each occurrence of P and Q is, individually, an abasic monomer asdefined above or a nucleotide, such as A, C, G, U or an analogue orequivalent thereof and p and q are each individually an integer,preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or higher up to 100. Thus, p and q are each individually aninteger from 0 to 100, preferably an integer from 0 to 20, morepreferably an integer from 0 to 10, more preferably from 0 to 6, evenmore preferably from 0 to 3. Thus, when p is 0, P is absent and when qis 0, Q is absent. The skilled person will appreciate that anoligonucleotide will always start with and end with a hydrogen atom (H),regardless of the amount and nature of the nucleotides present in theoligonucleotide.

It will be appreciated that herein (CUG)_(m) may be replaced by anyrepeating nucleotide unit within the context of the invention. Thus, apreferred oligonucleotide according to the invention may be representedby H-(P)_(p)—(R)_(r)-(Q)_(q)-H, wherein (R)_(r) is a repeatingnucleotide unit within the context of the invention and P, Q, p and qare as defined above.

In the context of the present invention, an “analogue” or an“equivalent” of a nucleotide is to be understood as a nucleotide whichcomprises at least one modification with respect to the nucleotidesnaturally occurring in RNA, such as A, C, G and U. Such a modificationmay be a intemucleoside linkage modification and/or a sugar modificationand/or a base modification, as explained and exemplified above.

Again taking the oligonucleotide having CUG as repetitive nucleotideunit, it is to be understood that the repeating sequence may start witheither a C, a U or a G. Thus, in a preferred embodiment, p is not 0, and(P)_(p) is represented by (P′)_(p′)UG or (P′)_(p″)G, wherein eachoccurrence of P′ is, individually, an abasic site or a nucleotide, suchas A, C, G, U or an analogue or equivalent thereof, and p′ is p−2 and p″is p−1. Such oligonucleotides may be represented as:H-(P′)_(p′)UG-(CUG)_(m)-(Q)_(q)-H orH-(P′)_(p″)G-(CUG)_(m)-(Q)_(q)-H.

In an equally preferred embodiment, q is not 0, and (Q)_(q) isrepresented by CU(Q′)_(q′) or C(Q′)_(q″) and each occurrence of Q′ is,individually, an abasic site or a nucleotide, such as A, C, G, U or ananalogue or equivalent thereof, and q′ is q−2 and q″ is q−1. Sucholigonucleotides may be represented as:H-(P)_(p)-(CUG)_(m)-CU(Q′)_(q′)-H orH-(P)_(p)-(CUG)_(m)-C(Q′)_(q″)-H.

In another preferred embodiment, both p and q are not 0, and both(P)_(p) and (Q)_(q) are represented by (P′)_(p′)UG or (P′)_(p″)G andCU(Q′)_(q′) or C(Q′)_(q″) respectively, wherein P′, Q′, p′, p″, q′ andq″ are as defined above. Such oligonucleotides may be represented as:H-(P′)_(p′)UG-(CUG)_(m)-CU(Q′)_(q′)-H,H-(P′)_(p″)G-(CUG)_(m)-CU(Q′)_(q′)-H,H-(P′)_(p′)UG-(CUG)_(m)-C(Q′)_(q″)-H, orH-(P′)_(p″)G-(CUG)_(m)-C(Q′)_(q″)-H.

It is to be understood that p′, p″, q′ and q″ may not be negativeintegers. Thus, when (P)_(p) is represented by (P′)_(p′)UG or(P′)_(p″)G, p is at least 1 or at least 2 respectively, and when (Q)_(q)is represented by CU(Q′)_(q′) or C(Q′)_(q″), q is at least 1 or at least2 respectively. It is to be understood that all said here regarding theCUG repeat unit can be extended to any repeat unit within the context ofthe invention.

In a preferred embodiment, an oligonucleotide defined as being reversecomplementary to, binding (or being able to bind), hybridizing (or beingable to hybridize) or targeting a (CAG)_(n) repeat comprises or consistsof a repetitive nucleotide unit (XYG)_(m) and has a length comprisedfrom 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine,and each Y is U or 5-methyluracil such that at least one X is5-methylcytosine and/or at least one Y is 5-methyluracil. m is aninteger. In the context of this embodiment, m may be 4, 5, 6, 7, 8, 9,10, 11, 12. A preferred value for m is 7.

A more preferred oligonucleotide therefore comprises or consists of arepetitive nucleotide unit (XYG)_(m), wherein each X is C or5-methylcytosine, and each Y is U or 5-methyluracil such that at leastone X is 5-methylcytosine and/or at least one Y is 5-methyluracil, and mis an integer from 4 to 12 (SEQ ID NO:2 to 12).

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XYG)_(m), wherein each X is5-methylcytosine, and/or each Y is 5-methyluracil, and m is an integerfrom 4 to 12 (SEQ ID NO:2 to 12).

An even more preferred oligonucleotide therefore comprises or consistsof a repetitive nucleotide unit (XYG)₅, (XYG)₆ or (XYG)₇, (XYG)₈, or(XYG)₉ wherein each X is C or 5-methylcytosine, and each Y is U or5-methyluracil such that at least one X is 5-methylcytosine and/or atleast one Y is 5-methyluracil. More preferred is an oligonucleotidecomprising or consisting of (XYG)₇, wherein each X is C or5-methylcytosine, and each Y is U or 5-methyluracil such that at leastone X is 5-methylcytosine and/or at least one Y is 5-methyluracil (SEQID NO:7).

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XYG)₇, wherein each X is 5-methylcytosineand each Y is a uracil (SEQ ID NO: 2), or each X is a cytosine and eachY is 5-methyluracil (SEQ ID NO:3). An even more preferredoligonucleotide comprises SEQ ID NO:2 or 3 and has a length of 21, 22,23, 24, 25, 26, 27, 28, 29, 30 nucleotides.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (XYG)_(m) have been identified in table 2 asSEQ ID NO:90-118.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 90-106and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An evenmore preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA and comprises one of the base sequences SEQ ID NO: 90-106 and has alength from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a basesequence that consists of one of the base sequences SEQ ID NO: 90-106and has a length of 21 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 107 or108 and has a length from 21-36 nucleotides, more preferably 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:107 or 108 and has a length from 21-36 nucleotides, more preferably 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. Even more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA, has a base sequence that consists of one of thebase sequences SEQ ID NO: 107 or 108 and has a length of 21 nucleotides.

Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 107 or 108, has a length of 21 nucleotides and additionallycomprises 4 abasic monomers at one of its termini, preferably at the 3′terminus. Said most preferred oligonucleotide is represented by a basesequence consisting of SEQ ID NO: 220 or 221.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 109 or110 and has a length from 24-36 nucleotides, more preferably 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even morepreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNAand comprises one of the base sequences SEQ ID NO: 109 or 110 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 109 or 110 and has alength of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 111 or112 and has a length from 27-36 nucleotides, more preferably 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides.

An even more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:111 or 112 and has a length from 27-36 nucleotides, more preferably 27,28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a basesequence that consists of one of the base sequences SEQ ID NO: 111 or112 and has a length of 27 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 113 or114 and has a length from 30-36 nucleotides, more preferably 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 113 or 114 and has a length from 30-36nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 113 or 114 and has a length of 30 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 115 or116 and has a length from 33-36 nucleotides, more preferably 33, 34, 35or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 115 or 116 and has a length from 33-36 nucleotides, morepreferably 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 115 or 116 and has alength of 33 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 117 or118 and has a length of 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 117 or 118 and has alength of 36 nucleotides.

In another embodiment, an antisense oligonucleotide comprising orconsisting of 2′-O-methyl phosphorothioate RNA, and comprising a5-methylcytosine is represented by a nucleotide sequence comprising orconsisting of a sequence that binds to (or is able to bind to),hybridizes (or is able to hybridize), targets and/or is reversecomplementary to a (GCG)_(n) repeat in a transcript and is particularlyuseful for the treatment, delay, amelioration and/or prevention of thehuman genetic diseases: infantile spasm syndrome, deidocranialdysplasia, blepharophimosis, hand-foot-genital disease, synpolydactyly,oculopharyngeal muscular dystrophy and/or holoprosencephaly, which arecaused by repeat expansions in the ARX, CBFA1, FOXL2, HOXA13, HOXD13,OPDM/PABP2, TCFBR1 or ZIC2 genes. Preferably, these genes are from humanorigin.

In a preferred embodiment, an oligonucleotide defined as being reversecomplementary to, binding (or being able to bind), hybridizing (or beingable to hybridize) or targeting a (GCG)_(n) repeat comprises or consistsof a repetitive nucleotide unit (XGX)_(m) and has a length comprisedfrom 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine,such that at least one X is 5-methylcytosine. m is an integer. In thecontext of this embodiment, m may be 4, 5, 6, 7, 8, 9, 10, 11, 12. Apreferred value for m is 7.

A more preferred oligonucleotide therefore comprises or consists of arepetitive nucleotide unit (XGX)_(m), wherein at least one X is5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 13 to21). An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XGX)_(m), wherein each X is5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 13 to21).

An even more preferred oligonucleotide therefore comprises or consistsof a repetitive nucleotide unit (XGX)₇ (SEQ ID NO: 16), wherein at leastone X is 5-methylcytosine.

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XGX)₇ (SEQ ID NO: 16), wherein each X is5-methylcytosine.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (XGX)_(m) have been identified in table 2 asSEQ ID NO:119-132.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 119 or120 and has a length from 12-36 nucleotides, more preferably 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 119 or 120 and has a length from 16-36nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 119 or 120 and has a length of 12 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 121 or122 and has a length from 15-36 nucleotides, more preferably 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 90-106 and has a length from 15-36 nucleotides, morepreferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 121 or 122 and has alength of 15 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 123 or124 and has a length from 18-36 nucleotides, more preferably 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 123 or 124 and has a length from 18-36 nucleotides, morepreferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has a base sequence that consists ofone of the base sequences SEQ ID NO: 123 or 124 and has a length of 18nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 125 or126 and has a length from 21-36 nucleotides, more preferably 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:125 or 126 and has a length from 21-36 nucleotides, more preferably 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. Most preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA, has a base sequence that consists of one of thebase sequences SEQ ID NO: 125 or 126 and has a length of 21 nucleotides.A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 127 or128 and has a length from 24-36 nucleotides, more preferably 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even morepreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNAand comprises one of the base sequences SEQ ID NO: 127 or 128 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 127 or 128 and has alength of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 129 or130 and has a length from 27-36 nucleotides, more preferably 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides.

An even more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:129 or 130 and has a length from 27-36 nucleotides, more preferably 27,28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a basesequence that consists of one of the base sequences SEQ ID NO: 129 or130 and has a length of 27 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 131 or132 and has a length from 30-36 nucleotides, more preferably 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 131 or 132 and has a length from 30-36nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 131 or 132 and has a length of 30 nucleotides.

In another embodiment, an oligonucleotide comprising or consisting of2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosine, isrepresented by a nucleotide sequence comprising or consisting of asequence that binds (or is able to bind), targets, hybridizes (or isable to hybridize) and/or is reverse complementary to a (CGG)_(n) repeatin a transcript and is particularly useful for the treatment, delay,amelioration and/or prevention of human fragile X syndromes, caused byrepeat expansion in the FMR1 gene. Preferably, these genes are fromhuman origin.

In a preferred embodiment, an oligonucleotide defined as being reversecomplementary to, binding (or is able to bind), hybridizing (or is ableto hybridize) or targeting a (CGG)_(n) repeat comprises or consists of arepetitive nucleotide unit (XXG)_(m) and has a length comprised from 12to 36 nucleotides and wherein each X is C or 5-methylcytosine, such thatat least one X is 5-methylcytosine.

m is an integer. In the context of this embodiment, m may be 4, 5, 6, 7,8, 9, 10, 11, 12.

A preferred value for m is 7.

A more preferred oligonucleotide therefore comprises or consists of arepetitive nucleotide unit (XXG)_(m), wherein each X is C or5-methylcytosine, such that at least one X is 5-methylcytosine, and m isan integer from 4 to 12 (SEQ ID NO: 22 to 30).

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XXG)_(m), wherein each X is5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 22 to30).

An even more preferred oligonucleotide therefore comprises or consistsof a repetitive nucleotide unit (XXG)₇ (SEQ ID NO: 25), wherein each Xis C or 5-methylcytosine, such that at least one X is 5-methylcytosine.

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XXG)₇ (SEQ ID NO: 25), wherein each X is5-methylcytosine.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (XXG)_(m) have been identified in table 2 asSEQ ID NO: 133-146.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 133 or134 and has a length from 12-36 nucleotides, more preferably 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 133 or 134 and has a length from 12-36nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 133 or 134 and has a length of 12 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 135 or136 and has a length from 15-36 nucleotides, more preferably 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 135 or 136 and has a length from 15-36 nucleotides, morepreferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 135 or 136 and has alength of 15 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 137 or138 and has a length from 18-36 nucleotides, more preferably 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 137 or 138 and has a length from 18-36 nucleotides, morepreferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has a base sequence that consists ofone of the base sequences SEQ ID NO: 137 or 138 and has a length of 18nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 139 or140 and has a length from 21-36 nucleotides, more preferably 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:139 or 140 and has a length from 21-36 nucleotides, more preferably 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. Most preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA, has a base sequence that consists of one of thebase sequences SEQ ID NO: 139 or 140 and has a length of 21 nucleotides.A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 141 or142 and has a length from 24-36 nucleotides, more preferably 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even morepreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNAand comprises one of the base sequences SEQ ID NO: 141 or 142 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 141 or 142 and has alength of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 143 or144 and has a length from 27-36 nucleotides, more preferably 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides.

An even more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:143 or 144 and has a length from 27-36 nucleotides, more preferably 27,28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a basesequence that consists of one of the base sequences SEQ ID NO: 143 or144 and has a length of 27 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 145 or146 and has a length from 30-36 nucleotides, more preferably 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 145 or 146 and has a length from 30-36nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 145 or 146 and has a length of 30 nucleotides.

In another embodiment, an oligonucleotide comprising or consisting of2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosineand/or a 5-methyluracil, is represented by a nucleotide sequencecomprising or consisting of a sequence that binds (or is able to bind),targets, hybridizes (or is able to hybridize) and/or is reversecomplementary to a (GAA)_(n) repeat in a transcript and is particularlyuseful for the treatment, delay and/or prevention of the human geneticdisorder Friedreich's ataxia.

In a preferred embodiment, an oligonucleotide defined as being reversecomplementary to, binding (or being able to bind), hybridizing (or beingable to hybridize) or targeting a (GAA)_(n) repeat comprises or consistsof a repetitive nucleotide unit (YYX)_(m) and has a length comprisedfrom 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine,and each Y is U or 5-methyluracil such that at least one X is5-methylcytosine and/or at least one Y is 5-methyluracil. m is aninteger. In the context of this embodiment, m may be 4, 5, 6, 7, 8, 9,10, 11, 12.

A preferred value for m is 7.

A more preferred oligonucleotide therefore comprises or consists of arepetitive nucleotide unit (YYX)_(m), wherein each X is C or5-methylcytosine, and each Y is U or 5-methyluracil such that at leastone X is 5-methylcytosine and/or at least one Y is 5-methyluracil, and mis an integer from 4 to 12 (SEQ ID NO: 31 to 39).

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (YYX)_(m), wherein each X is5-methylcytosine, and/or each Y is 5-methyluracil, and m is an integerfrom 4 to 12 (SEQ ID NO: 31 to 39).

An even more preferred oligonucleotide therefore comprises or consistsof a repetitive nucleotide unit (YYX)₇ (SEQ ID NO: 34), wherein each Xis C or 5-methylcytosine, and each Y is U or 5-methyluracil such that atleast one X is 5-methylcytosine and/or at least one Y is 5-methyluracil.

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (YYX)₇ (SEQ ID NO: 34), wherein each X is5-methylcytosine, and/or each Y is 5-methyluracil.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (XYG)_(m) have been identified in table 2 asSEQ ID NO: 147-167.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 147 or148 and has a length from 12-36 nucleotides, more preferably 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 147 or 148 and has a length from 12-36nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 147 or 148 and has a length of 12 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 149 or150 and has a length from 15-36 nucleotides, more preferably 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 149 or 150 and has a length from 15-36 nucleotides, morepreferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 149 or 150 and has alength of 15 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 151 or152 and has a length from 18-36 nucleotides, more preferably 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 151 or 152 and has a length from 18-36 nucleotides, morepreferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has a base sequence that consists ofone of the base sequences SEQ ID NO: 151 or 152 and has a length of 18nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 153-157and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An evenmore preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA and comprises one of the base sequences SEQ ID NO: 153-157 and has alength from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a basesequence that consists of one of the base sequences SEQ ID NO: 153-157and has a length of 21 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 158 or159 and has a length from 24-36 nucleotides, more preferably 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even morepreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNAand comprises one of the base sequences SEQ ID NO: 158 or 159 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 158 or 159 and has alength of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 160 or161 and has a length from 27-36 nucleotides, more preferably 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides.

An even more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:160 or 161 and has a length from 27-36 nucleotides, more preferably 27,28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a basesequence that consists of one of the base sequences SEQ ID NO: 160 or161 and has a length of 27 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 162 or163 and has a length from 30-36 nucleotides, more preferably 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 162 or 163 and has a length from 30-36nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has a base sequence that consists of one of the base sequences SEQID NO: 162 or 163 and has a length of 30 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 164 or165 and has a length from 33-36 nucleotides, more preferably 33, 34, 35or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 164 or 165 and has a length from 33-36 nucleotides, morepreferably 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has a base sequence thatconsists of one of the base sequences SEQ ID NO: 164 or 165 and has alength of 33 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 166 or167 and has a length of 36 nucleotides. An even more preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA and has abase sequence that consists one of the base sequences SEQ ID NO: 166 or167 and has a length of 36 nucleotides.

In another embodiment, an antisense oligonucleotide comprising orconsisting of 2′-O-methyl phosphorothioate RNA and comprising a5-methylcytosine, is represented by a nucleotide sequence comprising orconsisting of a sequence that binds to (or is able to bind), hybridizes(or is able to hybridize), targets and/or is reverse complementary to a(CCG)_(n) or (GCC)_(n) repeat in a transcript and is particularly usefulfor the treatment, delay, amelioration and/or prevention of the humangenetic disorder fragile XE mental retardation, caused by repeatexpansion in the FMR2 gene. Preferably, these genes are from humanorigin.

In a preferred embodiment, an oligonucleotide defined as being reversecomplementary to, binding (or being able to bind), hybridizing (or beingable to hybridize) or targeting a (CCG)_(n) repeat comprises or consistsof a repetitive nucleotide unit (XGG)_(m) or (GGX)_(m) and has a lengthcomprised from 12 to 36 nucleotides and wherein each X is C or5-methylcytosine. m is an integer. In the context of this embodiment, mmay be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value for m is 7.

A more preferred oligonucleotide therefore comprises or consists of arepetitive nucleotide unit (XGG)_(m) or (GGX)_(m), wherein each X is Cor 5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 49 to57) or (SEQ ID NO: 40 to 48).

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XGG)_(m) or (GGX)_(m), wherein each X is5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 49 to 57)or (SEQ ID NO: 40 to 48).

An even more preferred oligonucleotide therefore comprises or consistsof a repetitive nucleotide unit (XGG)₇ (SEQ ID NO: 52) or (GGX)₇ (SEQ IDNO: 43), wherein each X is C or 5-methylcytosine.

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XGG)₇ (SEQ ID NO: 52) or (GGX)₇ (SEQ ID NO:43), wherein each X is 5-methylcytosine.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (GGX)_(m) have been identified in table 2 asSEQ ID NO: 168-177.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 168 and has alength from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequences SEQ ID NO:168 and has a length from 12-36 nucleotides, more preferably 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequences SEQ ID NO: 168 and has a length of 12 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 169 and has alength from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO:169 and has a length from 15-36 nucleotides, more preferably 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 169 and has a length of 15 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 170 and has alength from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises base sequence SEQ ID NO: 170 and hasa length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Mostpreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA,has its base sequence that consists of base sequence SEQ ID NO: 170 andhas a length of 18 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 171-174has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 171-174 and has a length from 21-36 nucleotides, morepreferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA, has its base sequence that consists of one of thebase sequences SEQ ID NO: 171-174 and has a length of 21 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 175 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA andcomprises base sequence SEQ ID NO: 175 and has a length from 24-36nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 175 and has a length of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 176 and has alength from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises base sequenceSEQ ID NO: 176 and has a length from 27-36 nucleotides, more preferably27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has itsbase sequence that consists of base sequence SEQ ID NO: 176 and has alength of 27 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 177 and has alength from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO:177 and has a length from 30-36 nucleotides, more preferably 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 177 and has a length of 30 nucleotides.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (XGG)_(m) have been identified in table 2 asSEQ ID NO: 178-184.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 178 and has alength from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO:178 and has a length from 12-36 nucleotides, more preferably 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 178 and has a length of 12 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 179 and has alength from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO:179 and has a length from 15-36 nucleotides, more preferably 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 179 and has a length of 15 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequences SEQ ID NO: 180 and has alength from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises base sequence SEQ ID NO: 180 and hasa length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Mostpreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA,has its base sequence that consists of base sequence SEQ ID NO: 180 andhas a length of 18 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 181 and has alength from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even morepreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNAand comprises base sequence SEQ ID NO: 181 and has a length from 21-36nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has its base sequence thatconsists of base sequence SEQ ID NO: 181 and has a length of 21nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 182 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA andcomprises base sequence SEQ ID NO: 182 and has a length from 24-36nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 182 and has a length of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 183 and has alength from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises base sequenceSEQ ID NO: 183 and has a length from 27-36 nucleotides, more preferably27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has itsbase sequence that consists of base sequence SEQ ID NO: 183 and has alength of 27 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 184 and has alength from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO:184 and has a length from 30-36 nucleotides, more preferably 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 184 and has a length of 30 nucleotides.

In another embodiment, an oligonucleotide comprising or consisting of2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosineand/or a 2,6-diaminopurine, is represented by a nucleotide sequencecomprising or consisting of a sequence that binds (or is able to bind),hybridizes (or is able to hybridize), targets and/or is reversecomplementary to a (CCUG)_(n) repeat in a transcript and is particularlyuseful for the treatment, delay and/or prevention of the human geneticdisorder myotonic dystrophy type 2 (DM2), caused by repeat expansions inthe DM2/ZNF9 gene. Preferably, these genes are from human origin.

In a preferred embodiment, an oligonucleotide defined as being reversecomplementary to, binding (or being able to bind), hybridizing (or beingable to hybridize) or targeting a (CCUG)_(n) repeat comprises orconsists of a repetitive nucleotide unit (XZGG)_(m) and has a lengthcomprised from 12 to 36 nucleotides and wherein each X is C or5-methylcytosine, and each Z is A or 2,6-diaminopurine such that atleast one X is 5-methylcytosine and/or at least one Z is2,6-diaminopurine.

m is an integer. In the context of this embodiment, m may be 3, 4, 5, 6,7, 8, 9. A preferred value for m is 5.

A more preferred oligonucleotide therefore comprises or consists of arepetitive nucleotide unit (XZGG)_(m), wherein each X is C or5-methylcytosine, and each Z is A or 2,6-diaminopurine such that atleast one X is 5methyl-cytosine and/or at least one A is2,6-diaminopurine, and m is an integer from 3 to 9 (SEQ ID NO: 63 to69).

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XZGG)_(m), wherein each X is5-methylcytosine, and/or each Z is 2,6-diaminopurine, and m is aninteger from 3 to 9 (SEQ ID NO: 63 to 69).

An even more preferred oligonucleotide therefore comprises or consistsof a repetitive nucleotide unit (XZGG)₅ (SEQ ID NO: 65), wherein each Xis C or 5-methylcytosine, and each Z is A or 2,6-diaminopurine such thatat least one X is 5-methylcytosine and/or at least one Z is2,6-diaminopurine.

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (XZGG)₅ (SEQ ID NO: 65), wherein each X is5-methyl-cytosine, and/or each Z is 2,6-diaminopurine.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (XZGG)_(m) have been identified in table 2 asSEQ ID NO: 193-208.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 193 or194 and has a length from 12-36 nucleotides, more preferably 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA and comprises one of thebase sequences SEQ ID NO: 193 or 194 and has a length from 12-36nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has its base sequence that consists of one of the base sequencesSEQ ID NO: 193 or 194 and has a length of 12 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 195 or196 and has a length from 16-36 nucleotides, more preferably 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 195 or 196 and has a length from 16-36 nucleotides, morepreferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has its base sequence thatconsists of one of the base sequences SEQ ID NO: 195 or 196 and has alength of 16 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 197-200and has a length from 20-36 nucleotides, more preferably 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:197-200 and has a length from 20-36 nucleotides, more preferably 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. Most preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA, has its base sequence that consists of one of thebase sequences SEQ ID NO: 197-200 and has a length of 20 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 201 or202 and has a length from 24-36 nucleotides, more preferably 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even morepreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNAand comprises one of the base sequences SEQ ID NO: 201 or 202 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has its base sequence thatconsists of one of the base sequences SEQ ID NO: 201 or 202 and has alength of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 203 or204 and has a length from 28-36 nucleotides, more preferably 28, 29, 30,31, 32, 33, 34, 35 or 36 nucleotides. An even more preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA andcomprises one of the base sequences SEQ ID NO: 203 or 204 and has alength from 28-36 nucleotides, more preferably 28, 29, 30, 31, 32, 33,34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofone of the base sequences SEQ ID NO: 203 or 204 and has a length of 28nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 205 or206 and has a length from 32-36 nucleotides, more preferably 32, 33, 34,35 or 36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 205 or 206 and has a length from 32-36 nucleotides, morepreferably 32, 33, 34, 35 or 36 nucleotides. Most preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has itsbase sequence that consists of one of the base sequences SEQ ID NO: 205or 206 and has a length of 32 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 207 or208 and has a length of 36 nucleotides. An even more preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA and has itsbase sequence that consists of one of the base sequences SEQ ID NO: 207or 208 and has a length of 36 nucleotides.

In another embodiment, an oligonucleotide comprising or consisting of2′-O-methyl phosphorothioate RNA and comprising a 5-methyluracil and/ora 2,6-diaminopurine, is represented by a nucleotide sequence comprisingor consisting of a sequence that binds (or is able to bind), hybridizes(or is able to hybridize), targets and/or is reverse complementary to a(AUUCU)_(n) repeat in an intron and is particularly useful for thetreatment, delay, amelioration and/or prevention of the human geneticdisorder spinocerebellar ataxia type 10 (SCA10). Preferably, this geneis from human origin. In a preferred embodiment, an oligonucleotidedefined as being reverse complementary to, binding (or being able tobind), hybridizing (or being able to hybridize) or targeting a(AUUCU)_(n) repeat comprises or consists of a repetitive nucleotide unit(ZGZZY)_(m) and has a length comprised from 12 to 36 nucleotides andwherein each Y is U or 5-methyluracil, and each Z is A or2,6-diaminopurine such that at least one Y is 5-methyluracil and/or atleast one Z is 2,6-diaminopurine.

m is an integer. In the context of this embodiment, m may be 3, 4, 5, 6,7. A preferred value for m is 4.

A more preferred oligonucleotide therefore comprises or consists of arepetitive nucleotide unit (ZGZZY)_(m), wherein each Y is U or5-methyluracil, and each Z is A or 2,6-diaminopurine such that at leastone Y is 5-methyluracil and/or at least one Z is 2,6-diaminopurine, andm is an integer from 3 to 7 (SEQ ID NO: 58 to 62).

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (ZGZZY)_(m), wherein each Y is5-methyluracil, and/or each Z is 2,6-diaminopurine, and m is an integerfrom 3 to 7 (SEQ ID NO: 58 to 62).

An even more preferred oligonucleotide therefore comprises or consistsof a repetitive nucleotide unit (ZGZZY)₄ (SEQ ID NO: 59), wherein each Yis C or 5-methyluracil, and each Z is A or 2,6-diaminopurine such thatat least one Y is 5-methyluracil and/or at least one Z is2,6-diaminopurine.

An even more preferred oligonucleotide comprises or consists of arepetitive nucleotide unit (ZGZZY)₄ (SEQ ID NO: 59), wherein each Y is5-methyluracil, and/or each Z is 2,6-diaminopurine.

Most preferred oligonucleotides sequences comprising or consisting of arepetitive nucleotide unit (ZGZZY)_(m) have been identified in table 2as SEQ ID NO:185-192.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 185 and has alength from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO:185 and has a length from 15-36 nucleotides, more preferably 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 185 and has a length of 15 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 186-189and has a length from 20-36 nucleotides, more preferably 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises one of the base sequences SEQ ID NO:186-189 and has a length from 20-36 nucleotides, more preferably 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. Most preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA, has its base sequence that consists of one of thebase sequences SEQ ID NO: 186-189 and has a length of 20 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 190 and has alength from 25-36 nucleotides, more preferably 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35 or 36 nucleotides.

An even more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises base sequence SEQ ID NO: 190 and hasa length from 25-36 nucleotides, more preferably 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has its base sequence thatconsists of base sequence SEQ ID NO: 190 and has a length of 25nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 191 and has alength from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or36 nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO:191 and has a length from 30-36 nucleotides, more preferably 30, 31, 32,33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequence SEQ ID NO: 191 and has a length of 30 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 192 and has alength from 35-36 nucleotides, more preferably 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises base sequence SEQ ID NO: 192 and hasa length from 35-36 nucleotides, more preferably 35 or 36 nucleotides.Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioateRNA, has its base sequence that consists of base sequence SEQ ID NO: 192and has a length of 35 nucleotides.

In another embodiment, an oligonucleotide comprising or consisting of2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosineand/or a abasic monomer, and/or a inosine, is represented by anucleotide sequence comprising or consisting of a sequence that binds(or is able to bind), hybridizes (or is able to hybridize), targetsand/or is reverse complementary to a (GGGGCC)_(n) repeat present in aC9ORF72 human transcript and is particularly useful for the treatment,delay, amelioration and/or prevention of the human genetic disorderamylotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).Preferably, this gene is from human origin.

In a preferred embodiment, an oligonucleotide defined as being reversecomplementary to, binding (or being able to bind), hybridizing (or beingable to hybridize) or targeting a (GGGGCC)_(n) repeat comprises orconsists of a repetitive nucleotide unit (GGXUXX)_(m), (GGXQXX)_(m),(GGXIXX)_(m), or (GGCCUC)_(m), and has a length comprised from 17 to 36nucleotides and wherein each X is C or 5-methylcytosine such that atleast one X is 5-methylcytosine, wherein each Q is an abasic monomer,wherein each I is an inosine, and wherein m is an integer. In thecontext of this embodiment, m may be 3, 4, 5, 6, 7. A preferred valuefor m is 3 or 4.

More preferably, said oligonucleotide comprises or consists of arepetitive nucleotide unit SEQ ID NO: 216-219 as defined in table 1.Even more preferred oligonucleotides sequences comprising or consistingof a repetitive nucleotide unit (GGXUXX)_(m), (GGXQXX)_(m),(GGXIXX)_(m), or (GGCCUC)_(m), have been identified in table 2 as SEQ IDNO:209-215.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 209 or211 and has a length from 17-36 nucleotides, more preferably 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36nucleotides. An even more preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA and comprises one of the base sequencesSEQ ID NO: 209 or 211 and has a length from 17-36 nucleotides, morepreferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has its base sequence thatconsists of one of the base sequences SEQ ID NO: 209 or 211 and has alength of 17 or 18 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 210 and has alength from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Aneven more preferred oligonucleotide consists of 2′-O-methylphosphorothioate RNA and comprises base sequence SEQ ID NO: 210 and hasa length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Mostpreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA,has its base sequence that consists of base sequence SEQ ID NO: 210 andhas a length of 18 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises one of base sequences SEQ ID NO: 212 or215 and has a length from 24-36 nucleotides, more preferably 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even morepreferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNAand comprises one of the base sequences SEQ ID NO: 212 or 215 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotideconsists of 2′-O-methyl phosphorothioate RNA, has its base sequence thatconsists of one of the base sequences SEQ ID NO: 212 or 215 and has alength of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 213 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA andcomprises base sequence SEQ ID NO: 213 and has a length from 24-36nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequences SEQ ID NO: 213 and has a length of 24 nucleotides.

A preferred oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA comprises base sequence SEQ ID NO: 214 and has alength from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferredoligonucleotide consists of 2′-O-methyl phosphorothioate RNA andcomprises base sequence SEQ ID NO: 214 and has a length from 24-36nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 or 36 nucleotides. Most preferred oligonucleotide consists of2′-O-methyl phosphorothioate RNA, has its base sequence that consists ofbase sequences SEQ ID NO: 214 and has a length of 24 nucleotides.

In an embodiment, an oligonucleotide preferably comprises or consists of2′-O-methyl phosphorothioate RNA, comprises a 5-methylcytosine and/or a5-methyluracil and/or a 2,6-diaminopurine base, is represented by anucleotide sequence comprising or consisting of at least 12 to 36consecutive nucleotides, said oligonucleotide targeting, hybridizing (oris able to hybridize), binding (or is able to bind) and/or being reversecomplementary to a repeat as earlier defined herein More preferably,said nucleotide sequence comprising or consisting of at least 12 to 36nucleotides, even more preferably 15 to 24, and most preferably 20 or 21nucleotides. The length of said oligonucleotide may be 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, or 36 nucleotides. Said oligonucleotide may be reversecomplementary to and/or capable of hybridizing to and/or capable oftargeting and/or capable of binding to a repeat in a coding region of atranscript, preferably a polyglutamine (CAG)_(n) coding tract. Saidoligonucleotide may also be reverse complementary to and/or capable ofhybridizing to and/or capable of targeting and/or capable of binding toa non-coding region for instance 5′ or 3′ untranslated regions, orintronic sequences present in precursor RNA molecules.

In the context of the invention, the expression “capable of” may bereplaced with “is able to”.

In a second aspect, the present invention relates to an oligonucleotide,which comprises one or more abasic sites, as defined further below, atone or both termini. Preferably 1 to 10, more preferably 2, 3, 4, 5, 6,7, 8, 9 or 10 and most preferably 4 abasic sites are present at a singleterminus or at both termini of the oligonucleotide. One or more abasicsites may be present and both free termini of the oligonucleotide (5′and 3′), or at only one. The oligonucleotide according to this aspect ofthe invention preferably is represented by a nucleotide or a basesequence comprising or consisting of a sequence that binds (or is ableto bind), hybridizes (or is able to hybridize), targets and/or isreverse complementary to a repetitive element in a RNA transcriptselected from the (CAG)_(n), (GCG)_(n), (CGG)_(n), (GAA)_(n), (GCC)_(n),(CCG)_(n), (AUUCU)_(n), (GGGGCC)_(n) or (CCUG)_(n), as indicated above.Said oligonucleotide is preferably a single stranded oligonucleotide,and may further optionally comprise any of the modifications asdiscussed herein, such as one or more base modifications, sugarmodifications and/or backbone modifications, such as 5-methyl-C,5-methyl-U, 2,6-diaminopurine, 2′-O-methyl, phosphorothioate, andcombinations thereof. It is to be understood that in this aspect of theinvention, these modification are not compulsory.

The oligonucleotide according to this aspect of the invention,comprising one or more abasic sites at one or both termini has animproved parameter over the oligonucleotides without such abasic sites.In this context, parameters may include: binding affinity and/orkinetics, silencing activity, allelic selectivity, biostability,(intra-tissue) distribution, cellular uptake and/or trafficking, and/orimmunogenicity of said oligonucleotide, as explained earlier herein inconnection with the improved parameter of an oligonucleotide of theinvention of the first aspect. Each of the assays and definitionsprovided herein in connection with the improvement of a parameter of anoligonucleotide of the first aspect also hold for an oligonucleotide ofthe second aspect.

Below, an oligonucleotide comprising or consisting of 2′-O-methylphosphorothioate RNA, comprising a 5-methylcytosine and/or a5-methyluracil base and being represented by a nucleotide or a basesequence comprising (CUG)_(m) and thus binding to (or being able to bindto), hybridizing (or being able to hybridize), targeting and/or beingreverse complementary to (CAG)_(n) is taken as an example to furtherillustrate the invention. Similar parameters defined in the context ofsuch oligonucleotide could be defined by the skilled person for otheroligonucleotides falling under the scope of the invention and binding to(or being able to bind to), hybridizing (or being able to hydridize),targeting and/or being reverse complementary to other repeats asidentified herein. Other or similar symptoms may be identified by theskilled person concerning other diseases as identified herein.

In a preferred embodiment, in the context of the invention, anoligonucleotide as designed herein is able to delay and/or cure and/ortreat and/or prevent and/or ameliorate a human genetic disorder asHuntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6,7, 12 or 17, amyotrophic lateral sclerosis (ALS), frontotemporaldementia (FTD), X-linked spinal and bulbar muscular atrophy (SBMA)and/or dentatorubropallidoluysian atrophy (DRPLA) caused by CAG repeatexpansions in the transcripts of a HTT (SEQ ID NO: 80), ATXN1 (SEQ IDNO: 81), ATXN2 (SEQ ID NO: 82) ATXN3 (SEQ ID NO: 83), CACNA1A (SEQ IDNO: 84), ATXN7 (SEQ ID NO: 85), PPP2R2B (SEQ ID NO: 86), TBP (SEQ ID NO:87), AR (SEQ ID NO: 88), ATN1 (SEQ ID NO: 89) genes when thisoligonucleotide is able to reduce or decrease the amount of (toxic)transcript of a diseased allele of a HTT, ATXN1, ATXN2 ATXN3, CACNA1A,ATXN7, PPP2R2B, TBP, AR or ATN1 gene in a cell of a patient, in a tissueof a patient and/or in a patient. In an embodiment, said HTT, ATXN1,ATXN2 ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 genes are humangenes.

In the case of HD, an expanded CAG repeat region is present in exon 1 ofthe HTT gene in the genome of a patient. An expanded CAG repeat regionmay be defined herein as comprising a consecutive repetition of 38 to180 repetitive units comprising a CAG trinucleotide, in a transcribedsequence of the HTT gene In the case of SCA1, an expanded CAG repeatregion is present in exon 8 of the ATXN1 gene in the genome of apatient. An expanded CAG repeat region may be defined herein ascomprising a consecutive repetition of 41 to 83 repetitive unitscomprising a CAG trinucleotide, in a transcribed sequence of the ATXN1gene.

In the case of SCA2, an expanded CAG repeat region is present in exon 1of the ATXN2 gene in the genome of a patient. An expanded CAG repeatregion may be defined herein as comprising a consecutive repetition of32 to 200 repetitive units comprising a CAG trinucleotide in atranscribed sequence of the ATXN2 gene.

In the case of SCA3, an expanded CAG repeat region is present in exon 8of the ATXN3 gene in the genome of a patient. An expanded CAG repeatregion may be defined herein as comprising a consecutive repetition of52 to 86 repetitive units comprising a CAG trinucleotide in atranscribed sequence of the ATXN3 gene.

In the case of SCA6, an expanded CAG repeat region is present in exon 47of the CACNA1A gene in the genome of a patient. An expanded CAG repeatregion may be defined herein as comprising a consecutive repetition of20 to 33 repetitive units comprising a CAG trinucleotide in atranscribed sequence of the CACNA1A gene.

In the case of SCA7, an expanded CAG repeat region is present in exon 3of the ATXN7 gene in the genome of a patient. An expanded CAG repeatregion may be defined herein as comprising a consecutive repetition of36 to at least 460 repetitive units comprising a CAG trinucleotide in atranscribed sequence of the ATXN7 gene.

In the case of SCA12, an expanded CAG repeat region may be present inthe 5′ untranslated region (UTR), in an intron or within an open readingframe of the PPP2R2B gene in the genome of a patient. An expanded CAGrepeat region may be defined herein as comprising a consecutiverepetition of 66 to 78 repetitive units comprising a CAG trinucleotidein a transcribed sequence of the PPP2R2B gene.

In the case of SCA17, an expanded CAG repeat region is present in exon 3of the TBP gene in the genome of a patient. An expanded CAG repeatregion may be defined herein as comprising a consecutive repetition of45 to 66 repetitive units comprising a CAG trinucleotide in atranscribed sequence of the TBP gene.

In the case of ALS or FTD, an expanded CAG repeat region is present inexon 1 of the ATXN2 gene in the genome of a patient. An expanded CAGrepeat region may be defined herein as comprising a consecutiverepetition of 27 to 33 repetitive units comprising a CAG trinucleotidein a transcribed sequence of the ATXN2 gene.

In the case of ALS or FTD, an expanded GGGGCC repeat region is presentin the first intron of the C9ORF72 gene in the genome of a patient. Anexpanded GGGGCC repeat region may be defined herein as comprising aconsecutive repetition of >30 repetitive units comprising a GGGGCChexanucleotide in a transcribed sequence of the C9ORF72 gene.

In the case of SBMA, an expanded CAG repeat region is present in exon 1of the AR gene in the genome of a patient. An expanded CAG repeat regionmay be defined herein as comprising a consecutive repetition of 40repetitive units comprising a CAG trinucleotide in a transcribedsequence of the AR gene.

In the case of DRPLA, an expanded CAG repeat region is present in exon 5of the ATN1 gene in the genome of a patient. An expanded CAG repeatregion may be defined herein as comprising a consecutive repetition of49 to 88 repetitive units comprising a CAG trinucleotide in atranscribed sequence of the ATN1 gene.

Throughout the invention, the term CAG repeat may be replaced by(CAG)_(n), and vice versa, wherein n is an integer that may be 6 to 29when the repeat is present in exon 1 of the HTT transcript of a healthyindividual, 6 to 39 when the repeat is present in exon 8 of the ATXN1gene of a healthy individual, less than 31 when the repeat is present inexon 1 of the ATXN2 gene of a healthy individual, 12 to 40 when therepeat is present in exon 8 of the ATXN3 gene of a healthy individual,less than 18 when the repeat is present in exon 47 of the CACNA1A geneof a healthy individual, 4 to 17 when the repeat is present in exon 3 ofthe ATXN7 gene of a healthy individual, 7 to 28 when the repeat ispresent in the 5′UTR of the PPP2R2B gene of a healthy individual, 25 to42 when the repeat is present in exon 3 of the TBP gene of a healthyindividual, 13 to 31 when the repeat is present in exon 1 of the AR geneof a healthy individual, 12 to 40 when the repeat is present in exon 8of the ATXN3 gene of a healthy individual, or 6 to 35 when the repeat ispresent in exon 5 of the ATN1 gene of a healthy individual.

It preferably means that an oligonucleotide of the invention reduces adetectable amount of disease-associated or disease-causing or mutanttranscript containing an extending or unstable number of CAG repeats ina cell of said patient, in a tissue of said patient and/or in a patient.Alternatively or in combination with previous sentence, saidoligonucleotide may reduce the translation of said mutant transcript andthus the amount of mutant (toxic) protein. The reduction or decrease ofthe amount of expanded CAG repeat transcripts may be at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100% by comparison to the amount of expanded CAGrepeat transcripts before the treatment. Another parameter may be thedecrease in (CAG)_(n) transcript or of the quantity of said mutanttranscript. This may be of at least. 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% bycomparison to the quantity of said transcript detected at the onset ofthe treatment

The reduction or decrease may be assessed by Northern Blotting orQ-RT-PCR, preferably as carried out in the experimental part. Anoligonucleotide of the invention may first be tested in the cellularsystem as described in Example 1 in the experimental part.

Alternatively or in combination with previous preferred embodiment, inthe context of the invention, an oligonucleotide as designed herein isable to delay and/or cure and/or treat and/or prevent and/or amelioratea human genetic disorder as Huntington's disease (HD), spinocerebellarataxia (SCA) type 1, 2, 3, 6, 7, 12 or 17, amyotrophic lateral sclerosis(ALS), frontotemporal dementia (FTD), X-linked spinal and bulbarmuscular atrophy (SBMA) and/or dentatorubropallidoluysian atrophy(DRPLA) caused by CAG repeat expansions in the transcripts of the HTT,ATXN1, ATXN2 ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 genes whenthis oligonucleotide is able to alleviate one or more symptom(s) and/orcharacteristic(s) and/or to improve a parameter linked with orassociated with Huntington's disease (HD), spinocerebellar ataxia (SCA)type 1, 2, 3, 6, 7, 12 or 17, amyotrophic lateral sclerosis (ALS),frontotemporal dementia (FTD), X-linked spinal and bulbar muscularatrophy (SBMA) and/or dentatorubropallidoluysian atrophy (DRPLA) in anindividual. An oligonucleotide as defined herein is able to improve oneparameter or reduce a symptom or characteristic if after at least oneweek, one month, six month, one year or more of treatment using a doseof said oligonucleotide of the invention as identified herein saidparameter is said to have been improved or said symptom orcharacteristic is said to have been reduced.

Improvement in this context may mean that said parameter had beensignificantly changed towards a value of said parameter for a healthyperson and/or towards a value of said parameter that corresponds to thevalue of said parameter in the same individual at the onset of thetreatment.

Reduction or alleviation in this context may mean that said symptom orcharacteristic had been significantly changed towards the absence ofsaid symptom or characteristic which is characteristic for a healthyperson and/or towards a change of said symptom or characteristic thatcorresponds to the state of the same individual at the onset of thetreatment.

In this context, symptoms for Huntington's Disease are choreiformmovements, progressive dementia and psychiatric manifestations(depression, psychosis, etc.). Choreiform movements consist ofinvoluntary, rapid, irregular, jerky motor actions including facialtwitching or writhing and twitching of distal extremities, and latermore generalized forms that may impair gait (Ropper and Brown, 2005).Each of these symptoms may be assessed by the physician using known anddescribed methods. A preferred method is monitoring of total functionalcapacity (TFC), a validated scale or symptom progression regarding thethree main symptomatic areas of HD, measured by validated rating scales.These areas are specifically progression of motor signs, progression ofneuropsychiatric symptoms and progression of cognitive decline. Anotherpreferred scale therefore is the Unified HD Rating Scale (UHDRS;Huntington Study Group (Kieburtz K. et al. 1996; 11:136-142).

Huntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6,7, or 17, X-linked spinal and bulbar muscular atrophy (SBMA) anddentatorubropallidoluysian atrophy (DRPLA) are all caused by CAG tripletrepeat expansions in the coding region of the gene. Although the diseasecausing proteins in these diseases are different, in each case theresulting expanded stretch of glutamines results in a toxic-gain-offunction of the protein and this leads to neurodegeneration. Proteinaggregates are found in the nucleus and cytoplasm of cells, indicatingthat protein misfolding is a common feature of these disorders. A commonpreferred parameter is therefore (mutant) protein levels which can bedetermined by western blot analysis (Evers et al.), or the presence ofprotein aggregates in the nucleus and/or cytoplasm which can bemonitored by in situ hybridization. An improvement of a HD parameter maybe the decrease in the detection of the quantity or amount of proteinaggregate. Such decrease may be at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% by comparison to the quantity or amount of protein aggregate beforethe onset of the treatment.

In the context of HD, various other proteins have been found toco-localize with htt aggregates, i.e. TATA box binding protein (TBP),CREB binding protein (CBP) and several molecular chaperones (Huang etal.; Muchowski et al.; Roon-Mom et al.; Steffan et al.). Also manyaffected cellular processes have been identified in HD, such astranscriptional de-regulation, mitochondrial dysfunction, and impairedvesicle transport, which may provide alternative parameters for HD(Bauer et al., 2009; Ross et al.). An improvement of each of thesepossible alternative HD parameters (i.e. TATA box binding protein (TBP),CREB binding protein (CBP) and several molecular chaperones) may bedefined as for the improvement of protein aggregate as defined above.

Composition

In a second aspect, there is provided a composition comprising anoligonucleotide as described in the previous section entitled“Oligonucleotide”. This composition preferably comprises or consists ofor essentially consists of an oligonucleotide as described above.

As explained in the first aspect of the invention for ALS and FTD, it isknown that at least two distinct repeats in at least two distincttranscripts may be involved in, responsible for, or linked with thedisease. All preferred features relating to each of theseoligonucleotides have been disclosed in the section entitled“oligonucleotide”. In a preferred embodiment, said composition is foruse as a medicament. Said composition is therefore a pharmaceuticalcomposition. A pharmaceutical composition usually comprises apharmaceutically accepted carrier, diluent and/or excipient. In apreferred embodiment, a composition of the current invention comprises acompound as defined herein and optionally further comprises apharmaceutically acceptable formulation, filler, preservative,solubilizer, carrier, diluent, excipient, salt, adjuvant and/or solvent.Such pharmaceutically acceptable carrier, filler, preservative,solubilizer, diluent, salt, adjuvant, solvent and/or excipient may forinstance be found in Remington: The Science and Practice of Pharmacy,20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000. Thecompound as described in the invention possesses at least one ionizablegroup. An ionizable group may be a base or acid, and may be charged orneutral. An ionizable group may be present as ion pair with anappropriate counterion that carries opposite charge(s). Examples ofcationic counterions are sodium, potassium, cesium, Tris, lithium,calcium, magnesium, trialkylammonium, triethylammonium, andtetraalkylammonium. Examples of anionic counterions are chloride,bromide, iodide, lactate, mesylate, acetate, trifluoroacetate,dichloroacetate, and citrate. Examples of counterions have beendescribed [e.g. Kumar L. et al, 2008, which is incorporated here in itsentirety by reference].

A pharmaceutical composition may be further formulated to further aid inenhancing the stability, solubility, absorption, bioavailability,pharmacokinetics and cellular uptake of said compound, in particularformulations comprising excipients capable of forming complexes,nanoparticles, microparticles, nanotubes, nanogels, hydrogels,poloxamers or pluronics, polymersomes, colloids, microbubbles, vesicles,micelles, lipoplexes, and/or liposomes. Examples of nanoparticlesinclude polymeric nanoparticles, gold nanoparticles, magneticnanoparticles, silica nanoparticles, lipid nanoparticles, sugarparticles, protein nanoparticles and peptide nanoparticles.

A preferred composition comprises at least one excipient that mayfurther aid in enhancing the targeting and/or delivery of saidcomposition and/or said oligonucleotide to and/or into muscle and/orbrain tissue and/or to a neuronal tissue and/or a cell. A cell may be amuscular or a neuronal cell.

Many of these excipients are known in the art (e.g. see Bruno, 2011) andmay be categorized as a first type of excipient. Examples of first typeof excipients include polymers (e.g. polyethyleneimine (PEI),polypropyleneimine (PPI), dextran derivatives, butylcyanoacrylate(PBCA), hexylcyanoacrylate (PHCA), poly(lactic-co-glycolic acid) (PLGA),polyamines (e.g. spermine, spermidine, putrescine, cadaverine),chitosan, poly(amido amines) (PAMAM), poly(ester amine), polyvinylether, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG)cyclodextrins, hyaluronic acid, colominic acid, and derivativesthereof), dendrimers (e.g. poly(amidoamine)), lipids {e.g.1,2-dioleoyl-3-dimethylammonium propane (DODAP),dioleoyldimethylammonium chloride (DODAC), phosphatidylcholinederivatives [e.g 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)],lyso-phosphatidylcholine derivaties [e.g.1-stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-LysoPC)],sphingomyeline,2-{3-[bis-(3-amino-propyl)-amino]-propylamino}-N-ditetracedyl carbamoylmethylacetamide (RPR209120), phosphoglycerol derivatives [e.g.1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG-Na),phosphaticid acid derivatives [1,2-distearoyl-sn-glycero-3-phosphaticidacid, sodium salt (DSPA), phosphatidylethanolamine derivatives [e.g.dioleoyl-phosphatidylethanolamine (DOPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine(DSPE),2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE),],N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA),1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER),(1,2-dimyristyolxypropyl-3-dimethylhydroxy ethyl ammonium (DMRIE),(N1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN),dimethyldioctadecylammonium bromide (DDAB),1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC),(b-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-olelyl-amidetrihydrochloride (AtuFECT01), N,N-dimethyl-3-aminopropane derivatives[e.g. 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DoDMA),1,2-dilinoleyloxy-N,N-3-dimethylaminopropane (DLinDMA),2,2-dilinoleyl-4-dimethylaminomethyl [1,3]-dioxolane (DLin-K-DMA),phosphatidylserine derivatives[1,2-dioleyl-sn-glycero-3-phospho-L-serine, sodium salt (DOPS)],cholesterol}proteins (e.g. albumin, gelatins, atellocollagen), andpeptides (e.g. protamine, PepFects, NickFects, polyarginine, polylysine,CADY, MPG).

Another preferred composition may comprise at least one excipientcategorized as a second type of excipient. A second type of excipientmay comprise or contain a conjugate group as described herein to enhancetargeting and/or delivery of the composition and/or of theoligonucleotide of the invention to a tissue and/or cell and/or into atissue and/or cell, as for example muscle or neuronal tissue or cell.Both types of excipients may be combined together into one singlecomposition as identified herein.

The skilled person may select, combine and/or adapt one or more of theabove or other alternative excipients and delivery systems to formulateand deliver a compound for use in the present invention.

Such a pharmaceutical composition of the invention may be administeredin an effective concentration at set times to an animal, preferably amammal. More preferred mammal is a human being. An oligonucleotide or acomposition as defined herein for use according to the invention may besuitable for direct administration to a cell, tissue and/or an organ invivo of individuals affected by or at risk of developing a disease orcondition as identified herein, and may be administered directly invivo, ex vivo or in vitro. Administration may be via systemic and/orparenteral routes, for example intravenous, subcutaneous,intraventricular, intrathecal, intramuscular, intranasal, enteral,intravitreal, intracerebral, epidural or oral route.

Preferably, such a pharmaceutical composition of the invention may beencapsulated in the form of an emulsion, suspension, pill, tablet,capsule or soft-gel for oral delivery, or in the form of aerosol or drypowder for delivery to the respiratory tract and lungs. In an embodimentan oligonucleotide of the invention may be used together with anothercompound already known to be used for the treatment of said disease.Such other compounds may be used for slowing down progression ofdisease, for reducing abnormal behaviors or movements, for reducingmuscle tissue inflammation, for improving muscle fiber and/or neuronalfunction, integrity and/or survival and/or improve, increase or restorecardiac function. Examples are, but not limited to, a steroid,preferably a (gluco)corticosteroid, an ACE inhibitor (preferablyperindopril), an angiotensin II type 1 receptor blocker (preferablylosartan), a tumor necrosis factor-alpha (TNFα) inhibitor, a TGFβinhibitor (preferably decorin), human recombinant biglycan, a source ofmIGF-1, a myostatin inhibitor, mannose-6-phosphate, dantrolene,halofuginone, an antioxidant, an ion channel inhibitor, a proteaseinhibitor, a phosphodiesterase inhibitor (preferably a PDE5 inhibitor,such as sildenafil or tadalafil, and/or PDE10A inhibitors and/or MP-10),L-arginine, dopamine blockers, amantadine, tetrabenazine, co-enzyme Q10,antidepressants, anti-psychotics, anti-epileptics, mood-stabilizers ingeneral, omega-3-fatty acids, creatine monohydrate, KMO inhibitors(Kynurenine mono oxigenase) such as CHDI246, or HDAC4 inhibitors such asPBT2. Such combined use may be a sequential use: each component isadministered in a distinct composition. Alternatively each compound maybe used together in a single composition.

Use

In a further aspect, there is provided the use of a composition or anoligonucleotide as described in the previous sections for use as amedicament or part of therapy, or applications in which saidoligonucleotide exerts its activity intracellularly. Preferably, anoligonucleotide or composition of the invention is for use as amedicament or part of a therapy for preventing, delaying, curing,ameliorating and/or treating a human cis-element repeat instabilityassociated genetic disorder. A human cis-element repeat instabilityassociated genetic disorder is preferably a neuromuscular geneticdisorder, more preferably as identified earlier herein.

Method

In a further aspect, there is provided a method for preventing,treating, curing, ameliorating and/or delaying a condition or disease asdefined in the previous section in an individual, in a cell, tissue ororgan of said individual. The method comprising administering anoligonucleotide or a composition of the invention to said individual ora subject in the need thereof.

The method according to the invention wherein an oligonucleotide or acomposition as defined herein may be suitable for administration to acell, tissue and/or an organ in vivo of individuals affected by any ofthe herein defined diseases or at risk of developing said disease, andmay be administered in vivo, ex vivo or in vitro. An individual or asubject in need is preferably a mammal, more preferably a human being.

In a further aspect, there is provided a method for diagnosis whereinthe oligonucleotide of the invention is provided with a radioactivelabel or fluorescent label. In this method, an oligonucleotide of theinvention may be used as an in situ probe to detect foci (RNA/proteinaggregates resulting from the repeat expansion) in a sample from asubject. Said sample comprises cells from said subject.

In an embodiment, in a method of the invention, a concentration of anoligonucleotide or composition is ranged from 0.01 nM to 1 μM. Morepreferably, the concentration used is from 0.05 to 500 nM, or from 0.1to 500 nM, or from 0.02 to 500 nM, or from 0.05 to 500 nM, even morepreferably from 1 to 200 nM.

Dose ranges of an oligonucleotide or composition according to theinvention are preferably designed on the basis of rising dose studies inclinical trials (in vivo use) for which rigorous protocol requirementsexist. An oligonucleotide as defined herein may be used at a dose whichis ranged from 0.01 to 200 mg/kg or 0.05 to 100 mg/kg or 0.1 to 50 mg/kgor 0.1 to 20 mg/kg, preferably from 0.5 to 10 mg/kg.

Dose ranges of an oligonucleotide or composition according to theinvention may also be used at a dose which is

Ranged from 100 to 300 μg/week, 8 to 12 injections in total or

Ranged from 150 to 250 μg/week, 9 to 11 injections in total or

200 μg/week, 11 injections in total or

Ranged from 10 to 350 μg/day during two weeks or

Ranged from 50 to 250 μg/day during two weeks or

Ranged from 100 to 200 μg/day during two weeks or

Ranged from 20 to 80 μg/day during two weeks or

Ranged from 200 to 320 μg/day during two weeks or

320 μg/day, during two weeks or

30 μg/day, during two weeks.

The ranges of concentration or dose of oligonucleotide or composition asgiven above are preferred concentrations or doses for in vitro or exvivo uses. The skilled person will understand that depending on theidentity of the oligonucleotide used, the target cell to be treated, thegene target and its expression levels, the medium used and thetransfection and incubation conditions, the concentration or dose ofoligonucleotide used may further vary and may need to be optimised anyfurther.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. The verb “to comprise” is synonymous with the verb “tohave” unless otherwise indicated. In addition the verb “to consist” maybe replaced by “to consist essentially of” meaning that anoligonucleotide or a composition as defined herein may compriseadditional component(s) than the ones specifically identified, saidadditional component(s) not altering the unique characteristic of theinvention. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

Each embodiment as identified herein may be combined together unlessotherwise indicated. All patent and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

Definitions

Throughout the application, the word “binds”, “targets”, “hybridizes”could be used interchangeably when used in the context of an antisenseoligonucleotide which is reverse complementary to a part of a pre-mRNAas identified herein. In the context of the invention, “hybridizes” or“binds” is used under physiological conditions in a cell, preferably ahuman cell unless otherwise indicated.

As used herein, “hybridization” refers to the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymine, 5-methyluracil and uracil which pair through theformation of hydrogen bonds. The natural base guanine is nucleobasecomplementary to the natural bases cytosine and 5-methyl-cytosine.Hybridization can occur under varying circumstances. In particular,hybridization of an oligonucleotide of the invention with a targetedpre-mRNA can occur under varying circumstances. Similarly, binding of anoligonucleotide of the invention to a targeted pre-mRNA can occur undervarying circumstances. Preferably, said hybridization or said binding isassessed under physiological conditions in a cell, more preferably in ahuman cell. An oligonucleotide of the invention is preferably said to beable to bind to, or capable of binding to, or able to hybridize with, orcapable of hybridizing with, when said binding or hybridization occursunder physiological conditions in a cell, preferably a human cell.

As used herein, “nucleotide” refers to a nucleoside further comprising amodified or unmodified phosphate linking group or a non-phosphateinternucleoside linkage.

As used herein, “nucleotide analogue” or “nucleotide equivalent” refersto a nucleotide, which comprises at least one modification with respectto the nucleotides naturally occurring in RNA, such as A, C, G and U.Such a modification may be an internucleoside linkage modificationand/or a sugar modification and/or a base modification.

As used herein, “monomer” refers to a precursor in the synthesis of anoligomeric or polymeric compound. Also the monomeric unit or residuewithin such an oligomeric or polymeric compound is encompassed in theterm “monomer”. Thus, “monomer” and “nucleotide residue” may be usedinterchangeably throughout the description. Within the context of thepresent invention, a monomer is preferably a nucleotide. Preferredmonomers to be incorporated in the oligonucleotides according to theinvention are nucleotides comprising a 2′-O-methyl substituent, aphosphorothioate internucleoside linkage and a 5-methylpyrimidine and/ora 2,6-diaminopurine nucleobase.

As used herein, “nucleobase” refers to the heterocyclic base portion ofa nucleoside. Nucleobases may be naturally occurring or may be modifiedand therefore include, but are not limited to adenine, cytosine,guanine, uracil, thymine and analogues thereof such as5-methyl-cytosine. In certain embodiments, a nucleobase may comprise anyatom or group of atoms capable of hydrogen bonding to a base of anothernucleic acid. As used herein, “T_(m)” means melting temperature which isthe temperature at which the two strands of a duplex nucleic acidseparate. T_(m) is often used as a measure of duplex stability or thebinding affinity of an antisense compound toward a complementary RNAmolecule.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a pentose sugar comprising a substituent at the 2′ positionother than H or OH. 2′-modified nucleosides include, but are not limitedto, bicyclic nucleosides wherein the bridge connecting two carbon atomsof the sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′-substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O-CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), wherein each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-O-Me”, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” eachrefers to a nucleoside comprising a sugar comprising an —OCH₃ group atthe 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, the term “adenine analogue” means a chemically-modifiedpurine nucleobase that, when incorporated into an oligomer, is capableof forming a base pair with either a thymine or uracil of acomplementary strand of RNA or DNA. Preferably, such base pair is aWatson-Crick base pair, but analogues and slight deviations thereof arealso considered allowable within the context of the present invention.

As used herein, the term “uracil analogue” means a chemically-modifiedpyrimidine nucleobase that, when incorporated into an oligomer, iscapable of forming a base pair with either a adenine of a complementarystrand of RNA or DNA. Preferably, such base pair is a Watson-Crick basepair, but analogues and slight deviations thereof are also consideredallowable within the context of the present invention.

As used herein, the term “thymine analogue” means a chemically-modifiedpyrimidine nucleobase that, when incorporated into an oligomer, iscapable of forming a base pair with an adenine of a complementary strandof RNA or DNA. Preferably, such base pair is a Watson-Crick base pair,but analogues and slight deviations thereof are also consideredallowable within the context of the present invention.

As used herein, the term “cytosine analogue” means a chemically-modifiedpyrimidine nucleobase that, when incorporated into an oligomer, iscapable of forming a base pair with a guanine of a complementary strandof RNA or DNA. For example, cytosine analogue can be a 5-methylcytosine.Preferably, such base pair is a Watson-Crick base pair, but analoguesand slight deviations thereof are also considered allowable within thecontext of the present invention.

As used herein, the term “guanine analogue” means a chemically-modifiedpurine nucleobase that, when incorporated into an oligomer, is capableof forming a base pair with a cytosine of a complementary strand of RNAor DNA. Preferably, such base pair is a Watson-Crick base pair, butanalogues and slight deviations thereof are also considered allowablewithin the context of the present invention.

As used herein, the term “guanosine” refers to a nucleoside orsugar-modified nucleoside comprising a guanine or guanine analognucleobase.

As used herein, the term “uridine” refers to a nucleoside orsugar-modified nucleoside comprising a uracil or uracil analognucleobase.

As used herein, the term “thymidine” refers to a nucleoside orsugar-modified nucleoside comprising a thymine or thymine analognucleobase.

As used herein, the term “cytidine” refers to a nucleoside orsugar-modified nucleoside comprising a cytosine or cytosine analognucleobase.

As used herein, the term “adenosine” refers to a nucleoside orsugar-modified nucleoside comprising an adenine or adenine analognucleobase.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

As used herein, “internucleoside linkage” refers to a covalent linkagebetween adjacent nucleosides. An intemucleoside linkage may be anaturally occurring intemucleoside linkage, i.e. a 3′ to 5′phosphodiester linkage, or a modified intemucleoside linkage.

As used herein, “modified intemucleoside linkage” refers to anyintemucleoside linkage other than a naturally occurring intemucleosidelinkage.

As used herein, “backbone” refers to the chain of alternating sugarmoieties and intemucleoside linkages, as it occurs in anoligonucleotide. The oligonucleotide of the invention comprises at leastone phosphorodithioate intemucleoside linkage, but it has to beunderstood that more backbone modifications, such as sugar modificationsand/or internucleoside linkage modifications may be present in thebackbone.

As used herein, “oligomeric compound” refers to a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound is an oligonucleotide. In certain embodiments, anoligomeric compound is a single-stranded oligonucleotide. In certainembodiments, an oligomeric compound is a double-stranded duplexcomprising two oligonucleotides. In certain embodiments, an oligomericcompound is a single-stranded or double-stranded oligonucleotidecomprising one or more conjugate groups and/or terminal groups.

As used herein, “conjugate” refers to an atom or group of atoms bound toan oligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and clearance. Conjugate groups are routinely used in thechemical arts and are linked directly or via an optional linking moietyor linking group to the parent compound such as an oligomeric compound.In certain embodiments, conjugate groups includes without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes. In certain embodiments, conjugates areterminal groups. In certain embodiments, conjugates are attached to a 3′or 5′ terminal nucleoside or to an internal nucleoside of anoligonucleotide.

As used herein, “conjugate linking group” refers to any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound. Linking groups or bifunctional linking moieties such as thoseknown in the art are amenable to the present invention.

As used herein, “antisense compound” refers to an oligomeric compound,at least a portion of which is at least partially complementary to, orat least partially directed to, a target nucleic acid to which ithybridizes and modulates the activity, processing or expression of saidtarget nucleic acid.

As used herein, “expression” refers to the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, splicing, post-transcriptional modification, andtranslation.

As used herein, “antisense oligonucleotide” refers to an antisensecompound that is an oligonucleotide.

As used herein, “antisense activity” refers to any detectable and/ormeasurable activity attributable to the hybridization of an anti sensecompound to its target nucleic acid. In certain embodiments, suchactivity may be an increase or decrease in an amount of a nucleic acidor protein. In certain embodiments, such activity may be a change in theratio of splice variants of a nucleic acid or protein. Detection and/ormeasuring of antisense activity may be direct or indirect. In certainembodiments, antisense activity is assessed by observing a phenotypicchange in a cell or animal.

As used herein, “target nucleic acid” refers to any nucleic acidmolecule the expression, amount, or activity of which is capable ofbeing modulated by an antisense compound.

In certain embodiments, the target nucleic acid is DNA or RNA. Incertain embodiments, the target RNA is miRNA, mRNA, pre-mRNA, non-codingRNA, or natural antisense transcripts. For example, the target nucleicacid can be a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state,

As used herein, “target mRNA” refers to a pre-selected RNA molecule thatencodes a protein.

As used herein, “targeting” or “targeted to” refers to the associationof an antisense compound to a particular target nucleic acid molecule ora particular region of nucleotides within a target nucleic acidmolecule. An antisense compound targets a target nucleic acid if it issufficiently reverse complementary to the target nucleic acid to allowhybridization under physiological conditions. In this context“sufficiently reverse complementary” may be at least 90%, 95%, 97%, 99%or 100% reverse complementary with said targeted nucleic acid molecule.

As used herein, “target site” refers to a region of a target nucleicacid that is bound by an antisense compound. In certain embodiments, atarget site is at least partially within the 3′ untranslated region ofan RNA molecule. In certain embodiments, a target site is at leastpartially within the 5′ untranslated region of an RNA molecule. Incertain embodiments, a target site is at least partially within thecoding region of an RNA molecule. In certain embodiments, a target siteis at least partially within an exon of an RNA molecule. In certainembodiments, a target site is at least partially within an intron of anRNA molecule. In certain embodiments, a target site is at leastpartially within a miRNA target site of an RNA molecule. In certainembodiments, a target site is at least partially within a repeat regionof an RNA molecule.

As used herein, “target protein” refers to a protein, the expression ofwhich is modulated by an antisense compound. In certain embodiments, atarget protein is encoded by a target nucleic acid. In certainembodiments, expression of a target protein is otherwise influenced by atarget nucleic acid.

As used herein, “complementarity” in reference to nucleobases refers toa nucleobase that is capable of base pairing with another nucleobase.For example, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.Nucleobases comprising certain modifications may maintain the ability topair with a counterpart nucleobase and thus, are still capable ofnucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases refersto a pair of nucleobases that do not form hydrogen bonds with oneanother or otherwise support hybridization.

As used herein, “complementary” in reference to linked nucleosides,oligonucleotides, or nucleic acids, refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through nucleobase complementarity. In certain embodiments,an antisense compound and its target are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget).

Preferably the antisense compounds contain no more than about 15%, morepreferably not more than about 10%, most preferably not more than 5% orno mismatches. The remaining nucleotides are nucleobase complementary orotherwise do not disrupt hybridization (e.g., universal bases). One ofordinary skill in the art would recognize the compounds provided hereinare at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% complementary to atarget nucleic acid or reverse complementarity to a target nucleic acid.

As used herein, “modulation” refers to a perturbation of amount orquality of a function or activity when compared to the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As a further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing, resulting in a change in the amount of a particularsplice-variant present compared to conditions that were not perturbed.As a further example, modulation includes perturbing translation of aprotein.

As used herein, “motif” refers to a pattern of modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “the same modifications” refer to modifications relativeto naturally occurring molecules that are the same as one another,including absence of modifications. Thus, for example, two unmodifiedDNA nucleoside have “the same modification,” even though the DNAnucleoside is unmodified.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” refers to the modification of a nucleoside andincludes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “pharmaceutically acceptable salts” refers to salts ofactive compounds that retain the desired biological activity of theactive compound and do not impart undesired toxicological effectsthereto.

As used herein, the term “independently” means that each occurrence of arepetitive variable within a claimed oligonucleotide is selectedindependent of one another. For example, each repetitive variable can beselected so that (i) each of the repetitive variables are the same, (ii)two or more are the same, or (iii) each of the repetitive variables canbe different.

General Chemistry Definitions

As used herein, “alkyl” refers to a saturated straight or branchedhydrocarbon substituent or radical, typically containing up to twentyfour carbon atoms. Examples of alkyl groups include, but are not limitedto, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl,dodecyl and the like. Alkyl groups typically include from 1 to 24 carbonatoms, more typically from 1 to 12 carbon atoms (C₁-C₁₂ alkyl) with from1 to 6 carbon atoms (C₁-C₆ alkyl) being more preferred. The term “loweralkyl” as used herein includes from 1 to 6 carbon atoms (C₁-C₆ alkyl).Alkyl groups as used herein may optionally contain one or more furthersubstituents.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain radical or substituent, typically containing up to twenty fourcarbon atoms, and having at least one carbon-carbon double bond.Examples of alkenyl groups include, but are not limited to, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadienyland the like. Alkenyl groups typically include from 2 to 24 carbonatoms, more typically from 2 to 12 carbon atoms with from 2 to 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallycontain one or more further substituents.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonradical or substituent, typically containing up to twenty four carbonatoms, and having at least one carbon-carbon triple bond. Examples ofalkynyl groups include, but are not limited to, ethynyl, 1-propynyl,1-butynyl, and the like. Alkynyl groups typically include from 2 to 24carbon atoms, more typically from 2 to 12 carbon atoms with from 2 to 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally contain one or more further substituents.

As used herein, “aminoalkyl” refers to an amino substituted alkylradical or substituent. This term is meant to include C₁-C₁₂ alkylgroups having an amino substituent at any position and wherein theaminoalkyl group is attached to the parent molecule via its alkylmoiety. The alkyl and/or amino portions of the aminoalkyl group mayoptionally be further substituted with further substituents.

As used herein, “aliphatic” refers to a straight or branched hydrocarbonradical or substituent, typically containing up to twenty four carbonatoms, wherein the saturation between any two carbon atoms is a single,double or triple bond. An aliphatic group preferably contains from 1 to24 carbon atoms, more typically from 1 to 12 carbon atoms with from 1 to6 carbon atoms being more preferred. The straight or branched chain ofan aliphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally contain further substituents.

As used herein, “alicyclic” or “alicyclyl” refers to a cyclic radical orsubstituent, wherein the ring system is aliphatic. The ring system cancomprise one or more rings wherein at least one ring is aliphatic.Preferred alicyclic moieties include rings having from 5 to 9 carbonatoms in the ring. Alicyclic groups as used herein may optionallycontain further substituents.

As used herein, “alkoxy” refers to a radical or substituent comprisingan alkyl group and an oxygen atom, wherein the alkoxy group is attachedto a parent molecule via its oxygen atom. Examples of alkoxy groupsinclude, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy andthe like. Alkoxy groups as used herein may optionally contain furthersubstituents.

As used herein, “halo”, “halide” and “halogen” refer to an atom, radicalor substituent selected from fluorine, chlorine, bromine and iodine.

As used herein, “aryl” and “aromatic” refer to a radical or substituentcomprising a mono- or polycyclic carbocyclic ring system having one ormore aromatic rings.

Examples of aryl groups include, but are not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from 5 to 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally contain further substituents.

As used herein, “aralkyl” and “arylalkyl” refer to a radical orsubstituent comprising an alkyl group and an aryl group, wherein thearalkyl or arylalkyl group is attached to a parent molecule via itsalkyl moiety. Examples include, but are not limited to, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallycontain further substituents attached to the alkyl, the aryl or bothmoieties that form the radical or substituent.

As used herein, “heterocyclyl” refers to a radical or substituentcomprising a mono- or polycyclic ring system that includes at least oneheteroatom and is unsaturated, partially saturated or fully saturated,thereby including heteroaryl groups. Heterocyclyl is also meant toinclude fused ring system moieties wherein one or more of the fusedrings contain at least one heteroatom and the other rings can containone or more heteroatoms or optionally contain no heteroatoms. Aheterocyclic group typically includes at least one atom selected fromsulfur, nitrogen or oxygen. Examples of heterocyclic groups include[1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as usedherein may optionally contain further substituents. As used herein,“heteroaryl” and “heteroaromatic” refer to a radical or substituentcomprising a mono- or polycyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatom. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals or substituents can be attached to a parentmolecule directly or through a linking moiety such as an aliphatic groupor a heteroatom. Heteroaryl groups as used herein may optionally containfurther substituents.

As used herein, “heteroarylalkyl” refers to a radical or substituentcomprising a heteroaryl group as previously defined and an alkyl moiety,wherein the heteroarylalkyl group is attached to a parent molecule viaits alkyl moiety. Examples include, but are not limited to,pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the like.Heteroarylalkyl groups as used herein may optionally contain furthersubstituents on one or both of the heteroaryl or alkyl portions.

As used herein, “mono or polycyclic” refers to any ring systems, such asa single ring or a polycyclic system having rings that are fused orlinked, and is meant to be inclusive of single and mixed ring systemsindividually selected from aliphatic, alicyclic, aryl, heteroaryl,aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic andheteroarylalkyl. Such mono and polycyclic structures can contain ringsthat have a uniform or varying degree of saturation, including fullysaturated, partially saturated or fully unsaturated rings. Each ring cancomprise ring atoms selected from C, N, O and S to give rise toheterocyclic rings as well as rings comprising only C ring atoms.

Heterocyclic and all-carbon rings can be present in a mixed motif, suchas for example benzimidazole wherein one ring of the fused ring systemhas only carbon ring atoms and the other ring has two nitrogen atoms.The mono or polycyclic structures can be further substituted withsubstituents such as for example phthalimide which has two oxo groups(═O) attached to one of the rings. In another aspect, mono or polycyclicstructures can be attached to a parent molecule directly through a ringatom, through a substituent or a bifunctional linking moiety.

As used herein, “acyl” refers to a radical or substituent comprising acarbonyl moiety (C═O or —C(O)—) and a further substituent X, wherein theacyl group is attached to a parent molecule via its carbonyl moiety. Assuch, an acyl group is formally obtained by removal of a hydroxyl groupfrom an organic acid and has the general formula —C(O)—X, wherein X istypically aliphatic, alicyclic or aromatic. The term “acyl” is alsomeant to include heteroacyl radicals or substituents with generalformula —Y(O)_(n)—X, wherein X is as defined above and Y(O)_(n) istypically sulfonyl, sulfinyl or phosphate. Examples of acyl groupsinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallycontain further substituents.

As used herein, “substituent” and “substituent group” include groupsthat are typically added to other substituents or parent compounds toenhance desired properties or give desired effects. Substituent groupscan be protected or unprotected and can be attached to one availablesite or to many available sites in a parent compound. Substituent groupsmay also be further substituted with other substituent groups and may beattached directly or via a linking group such as an alkyl or hydrocarbylgroup to a parent compound. Herein, “hydrocarbyl” refers to any groupcomprising C, O and H. Included are straight, branched and cyclic groupshaving any degree of saturation. Such hydrocarbyl groups can include oneor more heteroatoms selected from N, O and S and can be furthersubstituted with one or more substituents.

Unless otherwise indicated, the term “substituted” or “optionallysubstituted” refers to the (optional) presence of any of the followingsubstituents: halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl(—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups, alicyclicgroups, alkoxy, substituted oxo (—O—R_(aa)), aryl, aralkyl,heterocyclic, heteroaryl, heteroarylalkyl, amino (—NR_(bb)R_(cc)), imino(═NR_(bb)), amido (—C(O)NR_(bb)R_(cc) or —N(R_(bb))C(O)R_(aa)), azido(—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)NR_(bb)R_(cc) or—N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)NR_(bb)R_(cc)), thioureido(—N(R_(bb))C(S)NR_(bb)R_(cc)), guanidinyl(—N(R_(bb))C(═NR_(bb))NR_(bb)R_(cc)), amidinyl(—C(═NR_(bb))NR_(bb)R_(cc) or —N(R_(bb))C(NR_(bb))R_(aa)), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)),sulfonamidyl (—S(O)₂NR_(bb)R_(cc) or —N(R_(bb))S(O)₂R_(bb)) andconjugate groups. Herein, each R_(aa), R_(bb) and R_(cc) is,independently, H, an optionally linked chemical functional group or afurther substituent, preferably but without limitation chosen from thegroup consisting of H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl,aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl.Selected substituents within the compounds described herein are presentto a recursive degree.

In this context, “recursive substituent” means that a substituent mayrecite another instance of itself. Because of the recursive nature ofsuch substituents, theoretically, a large number may be present in anygiven claim. One of ordinary skill in the art of medicinal chemistry andorganic chemistry understands that the total number of such substituentsis reasonably limited by the desired properties of the compoundintended. Such properties include, by way of example and not limitation,physical properties such as molecular weight, solubility or log P,application properties such as activity against the intended target andpractical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above.

As used herein, a zero (0) in a range indicating number of a particularunit means that the unit may be absent. For example, an oligomericcompound comprising 0-2 regions of a particular motif means that theoligomeric compound may comprise one or two such regions having theparticular motif, or the oligomeric compound may not have any regionshaving the particular motif. In instances where an internal portion of amolecule is absent, the portions flanking the absent portion are bounddirectly to one another. Likewise, the term “none” as used herein,indicates that a certain feature is not present. As used herein,“analogue” or “derivative” means either a compound or moiety similar instructure but different in respect to elemental composition from theparent compound regardless of how the compound is made. For example, ananalogue or derivative compound does not need to be made from the parentcompound as a chemical starting material.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. In vitro activity assay for (XYG)₇ in whichX=5-methylcytosine and Y=U (PS659 SEQ ID NO:90; derived from SEQ IDNO:2) and (XYG)₇ in which X=C and Y is 5-methyluracil (PS661 SEQ ID NO:97; derived from SEQ ID NO:3). PS659 (FIG. 1A) and PS661 (FIG. 1B) weretransfected into HD fibroblasts (GM04022) at increasing concentrations(0.5-200 nM). Efficacy and selectivity was determined with RT-PCR andlab-on-a-chip analysis. Silencing of the expanded ((CAG)₄₄) and healthy((CAG)₁₈) HTT transcripts were compared to the relative HTT transcriptlevels in mock samples. For all AONs n=2 except for mock (n=3).

FIGS. 2A-2D. In vivo efficacy of PS659 ((XYG)₇ in whichX=5-methylcytosine and Y=U; SEQ ID NO:2) in a transgenic HD rat model.Transgenic HD rats ((CAG)₅₁ repeat) received 15 times anintraventricular injection with PS659 (SEQ ID NO:90 derived from SEQ IDNO: 2), during 18 weeks at a final dose of 200 μg per injection, controlHD rats received vehicle only. Rats were sacrificed one week after thefinal injection. From all rats tissue was isolated and HTT levels weredetermined with Q-RT-PCR analysis. Reduced levels of HTT transcript werefound in (FIG. 2A) cortex, (FIG. 2B) hippocampus, (FIG. 2C) olfactorybulb and (FIG. 2D) thalamus after PS659 treatment compared to control.

TABLE 1 General structures of AONs. X = C or 5-methylcytosine Y = U or5-methyl- uracil, Z = A or 2,6-diaminopurine, I = inosine, and Q =abasic monomer. Target Repeat AON Sequence (5′ → 3′) SEQ ID NO (CAG)n(XYG)7 (PS57)  1 X = C, Y = U (XYG)7 (PS659)  2 X = 5-methylcytosine, Y= U (XYG)7 (PS661)  3 X = C, Y = 5-methyluracil (XYG)4  4 (XYG)5  5(XYG)6  6 (XYG)7  7 (XYG)8  8 (XYG)9  9 (XYG)10  10 (XYG)11  11 (XYG)12 12 (GCG)n (XGX)4  13 (XGX)5  14 (XGX)6  15 (XGX)7  16 (XGX)8  17 (XGX)9 18 (XGX)10  19 (XGX)11  20 (XGX)12  21 (CGG)n (XXG)4  22 (XXG)5  23(XXG)6  24 (XXG)7  25 (XXG)8  26 (XXG)9  27 (XXG)10  28 (XXG)11  29(XXG)12  30 (GAA)n (YYX)4  31 (YYX)5  32 (YYX)6  33 (YYX)7  34 (YYX)8 35 (YYX)9  36 (YYX)10  37 (YYX)11  38 (YYX)12  39 (GCC)n (GGX)4  40(GGX)5  41 (GGX)6  42 (GGX)7  43 (GGX)8  44 (GGX)9  45 (GGX)10  46(GGX)11  47 (GGX)12  48 (CCG)n (XGG)4  49 (XGG)5  50 (XGG)6  51 (XGG)7 52 (XGG)8  53 (XGG)9  54 (XGG)10  55 (XGG)11  56 (XGG)12  57 (AUUCU)n(ZGZZY)3  58 (ZGZZY)4  59 (ZGZZY)5  60 (ZGZZY)6  61 (ZGZZY)7  62 (CCUG)n(XZGG)3  63 (XZGG)4  64 (XZGG)5  65 (XZGG)6  66 (XZGG)7  67 (XZGG)8  68(XZGG)9  69 (GGGGCC)n (GGXUXX)3 216 (GGXUXX)4 217 (GGXIXX)4 218(GGXQXX)4 219 Note: All AONs with SEQ ID NO: 4-69, or 216-219 compriseat least one base modification selected from 5-methylcytosine,5-methyluracil, and 2,6-diaminopurine.

TABLE 2General structures of AONs. All AONs are 2′-O-methyl phosphorothioateAONs wherein C is 5-methylcytosine, U is 5-methyluracil, A is 2,6-diaminopurine, I is inosine and Q is an abasic monomer. Target AON SEQRepeat ID AON Sequence (5′→3′) ID NO (CAG)n PS659CUG CUG CUG CUG CUG CUG CUG  90 CUG CUG CUG CUG CUG CUG CUG  91CUG CUG CUG CUG CUG CUG CUG  92 CUG CUG CUG CUG CUG CUG CUG  93CUG CUG CUG CUG CUG CUG CUG  94 CUG CUG CUG CUG CUG CUG CUG  95CUG CUG CUG CUG CUG CUG CUG  96 PS661 CUG CUG CUG CUG CUG CUG CUG  97CUG CUG CUG CUG CUG CUG CUG  98 CUG CUG CUG CUG CUG CUG CUG  99CUG CUG CUG CUG CUG CUG CUG 100 CUG CUG CUG CUG CUG CUG CUG 101CUG CUG CUG CUG CUG CUG CUG 102 CUG CUG CUG CUG CUG CUG CUG 103 PS660CUG CUG CUG CUG CUG CUG CUG 104 CUG CUG CUG CUG CUG CUG CUG 105CUG CUG CUG CUG CUG CUG CUG 106 PS684 CUG CUG CUG CUG CUG CUG CUG 107CUG CUG CUG CUG CUG CUG CUG QQQQ 220 CUG CUG CUG CUG CUG CUG CUG 108CUG CUG CUG CUG CUG CUG CUG QQQQ 221 CUG CUG CUG CUG CUG CUG CUG CUG 109CUG CUG CUG CUG CUG CUG CUG CUG 110 CUG CUG CUG CUG CUG CUG CUG CUG CUG111 CUG CUG CUG CUG CUG CUG CUG CUG CUG 112CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 113CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 114CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 115 CUGCUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 116 CUGCUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 117 CUG CUGCUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 118 CUG CUG (GCG)nCGC CGC CGC CGC 119 CGC CGC CGC CGC 120 CGC CGC CGC CGC CGC 121CGC CGC CGC CGC CGC 122 CGC CGC CGC CGC CGC CGC 123CGC CGC CGC CGC CGC CGC 124 CGC CGC CGC CGC CGC CGC CGC 125CGC CGC CGC CGC CGC CGC CGC 126 CGC CGC CGC CGC CGC CGC CGC CGC 127CGC CGC CGC CGC CGC CGC CGC CGC 128 CGC CGC CGC CGC CGC CGC CGC CGC CGC129 CGC CGC CGC CGC CGC CGC CGC CGC CGC 130CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC 131CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC 132 (CGG)n CCG CCG CCG CCG 133CCG CCG CCG CCG 134 CCG CCG CCG CCG CCG 135 CCG CCG CCG CCG CCG 136CCG CCG CCG CCG CCG CCG 137 CCG CCG CCG CCG CCG CCG 138CCG CCG CCG CCG CCG CCG CCG 139 CCG CCG CCG CCG CCG CCG CCG 140CCG CCG CCG CCG CCG CCG CCG CCG 141 CCG CCG CCG CCG CCG CCG CCG CCG 142CCG CCG CCG CCG CCG CCG CCG CCG CCG 143CCG CCG CCG CCG CCG CCG CCG CCG CCG 144CCG CCG CCG CCG CCG CCG CCG CCG CCG CCG 145CCG CCG CCG CCG CCG CCG CCG CCG CCG CCG 146 (GAA)n UUC UUC UUC UUC 147UUC UUC UUC UUC 148 UUC UUC UUC UUC UUC 149 UUC UUC UUC UUC UUC 150UUC UUC UUC UUC UUC UUC 151 UUC UUC UUC UUC UUC UUC 152UUC UUC UUC UUC UUC UUC UUC 153 UUC UUC UUC UUC UUC UUC UUC 154UUC UUC UUC UUC UUC UUC UUC 155 UUC UUC UUC UUC UUC UUC UUC 156UUC UUC UUC UUC UUC UUC UUC 157 UUC UUC UUC UUC UUC UUC UUC UUC 158UUC UUC UUC UUC UUC UUC UUC UUC 159 UUC UUC UUC UUC UUC UUC UUC UUC UUC160 UUC UUC UUC UUC UUC UUC UUC UUC UUC 161UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 162UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 163UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 164 UUCUUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 165 UUCUUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 166 UUC UUCUUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 167 UUC UUC (GCC)nGGC GGC GGC GGC 168 GGC GGC GGC GGC GGC 169 GGC GGC GGC GGC GGC GGC 170GGC GGC GGC GGC GGC GGC GGC 171 GGC GGC GGC GGC GGC GGC GGC 172GGC GGC GGC GGC GGC GGC GGC 173 GGC GGC GGC GGC GGC GGC GGC 174GGC GGC GGC GGC GGC GGC GGC GGC 175 GGC GGC GGC GGC GGC GGC GGC GGC GGC176 GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC 177 (CCG)n CGG CGG CGG CGG178 CGG CGG CGG CGG CGG 179 CGG CGG CGG CGG CGG CGG 180CGG CGG CGG CGG CGG CGG CGG 181 CGG CGG CGG CGG CGG CGG CGG CGG 182CGG CGG CGG CGG CGG CGG CGG CGG CGG 183CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG 184 (AUUCU)n AGAAU AGAAU AGAAU185 AGAAU AGAAU AGAAU AGAAU 186 AGAAU AGAAU AGAAU AGAAU 187AGAAU AGAAU AGAAU AGAAU 188 AGAAU AGAAU AGAAU AGAAU 189AGAAU AGAAU AGAAU AGAAU AGAAU 190 AGAAU AGAAU AGAAU AGAAU AGAAU AGAAU191 AGAAU AGAAU AGAAU AGAAU AGAAU AGAAU 192 AGAAU (CCUG)n CAGG CAGG CAGG193 CAGG CAGG CAGG 194 CAGG CAGG CAGG CAGG 195 CAGG CAGG CAGG CAGG 196CAGG CAGG CAGG CAGG CAGG 197 CAGG CAGG CAGG CAGG CAGG 198CAGG CAGG CAGG CAGG CAGG 199 CAGG CAGG CAGG CAGG CAGG 200CAGG CAGG CAGG CAGG CAGG CAGG 201 CAGG CAGG CAGG CAGG CAGG CAGG 202CAGG CAGG CAGG CAGG CAGG CAGG CAGG 203CAGG CAGG CAGG CAGG CAGG CAGG CAGG 204CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 205CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 206CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 207 CAGGCAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 208 CAGG (GGGGCC)n PS1252GGCUCC GGCUCC GGCUC 209 GGCQCC GGCQCC GGCQCC 210 GGCUCC GGCUCC GGCUCC211 GGCUCC GGCUCC GGCUCC GGCUCC 212 GGCQCC GGCQCC GGCQCC GGCQCC 213GGCICC GGCICC GGCICC GGCICC 214 GGCCUC GGCCUC GGCCUC GGCCUC 215

EXAMPLES Example 1 Introduction

The particular characteristics of a chosen antisense oligonucleotide(AON) chemistry may at least in part enhance binding affinity andstability, enhance activity, improve safety, and/or reduce cost of goodsby reducing length or improving synthesis and/or purificationprocedures. This example describes the comparative analysis of theactivity of AONs designed to target the expanded (CAG)_(n) repeat in HTTtranscripts in HD fibroblasts in vitro, and includes AONs with either5-methylcytosines (XYG)₇, wherein X is 5-methylcytosine and Y=U beingalso identified as SEQ ID NO:90 (and derived from SEQ ID NO:2), or5-methyluracils (XYG)₇, wherein X=C and Y=5-methyluracil being alsoidentified as SEQ ID NO: 97 (and derived from SEQ ID NO:3).

Materials and Methods

Cell Culture.

Patient derived HD fibroblasts (GM04022) (purchased from Coriell CellRepositories, Camden, USA) were cultured at 37° C. and 5% CO₂ in MinimalEssential Medium (MEM) (Gibco Invitrogen, Carlsbad, USA) with 15% heatinactivated Fetal Bovine Serum (FBS) (Clontech, Palo Alto USA), 1%Glutamax (Gibco) and 100 U/ml penicillin/streptomycin (P/S) (Gibco).

Oligonucleotides.

The AONs were fully 2′-O-methyl phosphorothioate modified: PS659;(XYG)₇, wherein X is 5-methylcytosine and Y=U being also identified asSEQ ID NO: 90 (and derived from SEQ ID NO:2), and PS661; (XYG)₇, whereinX=C and Y=5-methyluracil being also identified as SEQ ID NO:97 (andderived from SEQ ID NO:3).

Transfection.

Cells were transfected with AONs complexed with PEI (2 μL per μg AON, in0.15 M NaCl). AON-PEI complex was added in MEM medium with 5% FBS tocells to a final AON concentration varying from 0.5-200 nM. Fresh mediumwas supplemented after four hours and after 24 hours RNA was isolated.

RNA Isolation.

RNA from cultured cells was isolated using the Aurum Total RNA Mini Kit(Bio-Rad, Hercules, Calif.) according to the manufacturer's protocol.

RT-PCR and Lab-On-a-Chip Analysis.

Approximately 200 ng RNA was subjected to cDNA synthesis with randomhexamers using the SuperScript first-strand synthesis system(Invitrogen) in a total volume of 20 μL. PCR was performed with primersfor HTT (across the CAG repeat) and β-actin. The PCR program startedwith a 4 min initial denaturation at 95° C., followed by 35 cycles of 30sec denaturation at 94° C., 30 sec annealing at 60° C., 45 secelongation at 72° C., after which a final elongation step was performedat 72° C. for 7 min. Lab-on-a-Chip was performed on the Agilent 2100Bioanalyzer (Agilent Technologies, Waldbronn, Germany), using theAgilent DNA 1000 Kit. Expression levels were normalized for β-actinlevels and relative to transcript levels without transfection. Thefollowing primers were used:

HTT forward; (SEQ ID NO: 70) 5′-ATGGCGACCCTGGAAAAGCTGAT-3′ HTT reverse:(SEQ ID NO: 71) 5′-TGAGGCAGCAGCGGCTG-3′ β-actin forward; (SEQ ID NO: 72)5′-GGACTTCGAGCAAGAGATGG-3′ β-actin reverse; (SEQ ID NO: 73)5′-AGCACTGTGTTGGCGTACAG-3′

Results

Both PS659 (SEQ ID NO: 90 derived from SEQ ID NO:2) and PS661 (SEQ IDNO: 97 derived from SEQ ID NO:3) were highly effective and reduced theHTT transcripts in HD fibroblasts in a dose-dependent manner (FIGS.1A-1B). Both AONs also showed a preference for the allele with theexpanded CAG repeats. PS659 (SEQ ID NO: 90 derived from SEQ ID NO:2) wasmore effective and more allele-specific at lower concentrations(strongest effect at 5 nM) (FIG. 1A) than PS661 (SEQ ID NO: 97 derivedfrom SEQ ID NO:3) (strongest effect at 20 nM) (FIG. 1B).

Example 2 Introduction

PS659 (XYG)₇, wherein X is 5-methylcytosine and Y=U also identified asSEQ ID NO: 90 (derived from SEQ ID NO:2), was selected from in vitrostudies as most efficient and safe candidate. This example describes itsactivity in a transgenic HD rat model after a series of directintraventricular injections.

Materials and Methods

Animals.

Transgenic HD rats carry a truncated Huntington cDNA fragment with 51CAG repeats under the control of the native rat Huntington promoter. Theexpressed gene product is about 75 kDa, corresponding to 22% of thefull-length Huntington (cDNA position 324-2321, amino acid position1-709/825, corresponding to exon 1-16), under the control of 886 bp ofthe rat Huntington promoter (von Hörsten S. et al.). All animalexperiments were approved by the Institutional Animal Care and UseCommittees of the Maastricht University, Maastricht.

Oligonucleotides.

PS659 (XYG)₇, wherein X is 5-methylcytosine and Y=U also identified asSEQ ID NO: 90 (derived from SEQ ID NO:2), is a fully 2′-O-methylphosphorothioate modified AON.

In Vivo Treatment.

Transgenic HD rats received 15 times an intraventricular injection at afinal dose of 200 μg PS659 also identified as SEQ ID NO: 90 (derivedfrom SEQ ID NO:2) during 18 weeks. Control HD rats received vehicleonly. Rats were sacrificed one week after the final injection.

RNA Isolation.

RNA from brain tissue was isolated using RNA-Bee reagent (Tel Test,Inc). In brief, tissue samples were homogenized in MagNA Lyser greenbead tubes (Roche) by adding RNA-Bee (50 mg tissue/mL RNA-Bee) andhomogenizing using a MagNA Lyser instrument (Roche). Lysate wastransferred to a new tube, chloroform (SIGMA) was added (0.2 mL per mLRNA-Bee), mixed, incubated on ice for 5 minutes and centrifuged at13,000 rpm for 15 minutes at 4° C. The upper aqueous phase was collectedand an equal volume isopropanol (SIGMA) was added, followed by a 1 hourincubation period at 4° C. and centrifugation (13,000 rpm, 15 min, 4°C.). The RNA precipitate was washed with 70% (v/v) ethanol (BioSolve),air dried and dissolved in MilliQ.

Quantitative RT-PCR Analysis.

Approximately 200 ng was subjected to cDNA synthesis with randomhexamers using the SuperScript first-strand synthesis system(Invitrogen) in a total volume of 20 μL. 3 μL of 1/40 cDNA dilutionpreparation was subsequently used in a quantitative PCR analysisaccording to standard procedures in presence of iQ™ SYBR® Green Supermix(Bio-Rad). Quantitative PCR primers were designed based on NCBI databasesequence information. Product identity was confirmed by DNA sequencing.The signal for Rab2 and YWHAZ was used for normalization. The followingprimers were used:

Rat Htt-F; (SEQ ID NO: 74) 5′-CGCCGCCTCCTCAGCTTC-3′ Rat Htt-R;(SEQ ID NO: 75) 5′-GAGAGTTCCTTCTTTGGTCGGTGC-3′ Rab2-F; (SEQ ID NO: 76)5′-TGGGAAACAGATAAAACTCCAGA-3′ Rab2-R; (SEQ ID NO: 77)5′-AATATGACCTTGTGATAGAACGAAAG-3′ YWHAZ-F; (SEQ ID NO: 78)5′-AAATGAGCTGGTGCAGAAGG-3′ YWHAZ-R; (SEQ ID NO: 79)5′-GGCTGCCATGTCATCGTAT-3′

Results

PS659 (also identified as SEQ ID NO: 90 or derived from SEQ ID NO: 2)reduced transgenic Htt transcript levels in cortex (FIG. 2A),hippocampus (FIG. 2B), olfactory bulb (FIG. 2C) as well as in thalamus(FIG. 2D) when compared to saline treated rats. These resultsdemonstrate that PS659 (also identified as SEQ ID NO: 90 or derived fromSEQ ID NO; 2) is effective in vivo after direct intraventricularinjection.

LIST OF REFERENCES

-   Aartsma-Rus et al., Hum Mol Gen 2003; 12(8):907-14.-   Arai K et al. Bioorg. Med. Chem. 2011, 21, 6285-   Bauer et al., 2009; J Neurochem. 110:1737-65-   Braida C., et al, Human Molecular Genetics, 2010, vol 9: 1399-1412-   Bruno et al., Adv Drug Deliv Rev. 2011; 63(13):1210-26-   Diebold et al., 2006, Eur J Immunol; 36(12): 3256-67-   Evers et al. PLoS ONE 2011, 6 (9) e24308-   Huang et al., 1998 Somat Cell Molec Gen 24:217-33;-   Krieg A M. et al., Nature 1995; 374: 546-549.-   Krieg, A. M., Curr. Opin. Immunol. 2000; 12: 35-43.-   Kumar L, Pharm. Technol. 2008, 3, 128.-   Muchowski et al., 2002 PNAS 99: 727-32-   Mulders et al. PNAS 2009 106(33); p 13915-20-   Peacock H et al. J. Am. Chem. Soc. 2011, 133, 9200-   Popovic P J. et al. J of Immunol 2006; 177: 8701-8707.-   Roon-Mom et al., 2002 Mol Brain Res 109: 1-10-   Ropper A H. and Brown R H., 2005 Principles of neurology. 8^(th) Ed.    New York:-   McGraw-Hill, 2005.-   Ross et al., 2011; Lancet Neurol. 10:83-98-   Rigo, F, et al, 2012, Nature chemical biology, 8: 555-561.-   Steffan et al., 2000 PNAS 97: 6763-68-   von Horsten S. et al. Hum Mol Genet. 2003; 12(6):617-24-   Wagner, H., Adv. Immunol. 1999; 73: 329-368.-   Yu R Z., Anal Biochem 2002; 304: 19-25.

The invention claimed is:
 1. A method for treating, delaying, ameliorating and/or preventing a spinocerebellar ataxia (SCA), spinal and bulbar muscular atrophy (SBMA), or dentatorubral-pallidoluysian atrophy (DRPLA) in a subject, comprising providing to the subject an antisense oligonucleotide comprising a 2′-O-methyl RNA nucleotide residue, having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, and comprising a repetitive nucleotide unit (XYG)m, wherein m is an integer from 4 to 12 and each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil, wherein at least one X is 5-methylcytosine, wherein said oligonucleotide is able to hybridize to a (CAG)n repetitive nucleotide unit.
 2. The method of claim 1, wherein said oligonucleotide consists of a repetitive nucleotide unit (XYG)m, wherein m is an integer from 4 to 12 and each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil, wherein at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil.
 3. The method of claim 1, wherein said oligonucleotide is such that each X is 5-methylcytosine and/or each Y is 5-methyluracil.
 4. The method of claim 1, wherein said oligonucleotide is such that m is 5 or 6 or 7 or 8 or 9 or 10 or 11 or
 12. 5. The method of claim 4, wherein said oligonucleotide comprises a repetitive nucleotide unit (XYG)7, wherein each X is 5-methylcytosine and each Y is a uracil (SEQ ID NO: 2).
 6. The method of claim 4, wherein said oligonucleotide is such that m is
 7. 7. The method of claim 1, wherein the sequence of said oligonucleotide comprises any of SEQ ID NO: 90, 96, 104-109, 111, 113, 115, and
 117. 8. The method of claim 7, wherein the base sequence of said oligonucleotide comprises SEQ ID NO:90.
 9. The method of claim 1, wherein said oligonucleotide is a single-stranded oligonucleotide.
 10. The method of claim 1 wherein said oligonucleotide has a length of from 12 to 36 nucleotides.
 11. The method of claim 1, wherein said oligonucleotide remain in association to its target when there are up to 20% of mismatched nucleotides.
 12. The method of claim 1, wherein said oligonucleotide is at least 90% reverse complementary with said repetitive nucleotide unit.
 13. The method of claim 1, wherein said oligonucleotide has an improved parameter by comparison to a corresponding oligonucleotide comprising a 2′-O-methyl RNA nucleotide residue and having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, without a 5-methylcytosine, and/or a 5-methyluracil and/or a 2,6-diaminopurine.
 14. The method of claim 1, wherein said oligonucleotide reduces a detectable amount of mutant transcript and/or reduces the translation of said mutant transcript and thus the amount of mutant protein.
 15. The method of claim 14, wherein said reduction of mutant protein reduces protein aggregates in nucleus and/or cytoplasm.
 16. The method of claim 1, wherein said oligonucleotide is present in a composition.
 17. The method of claim 16, wherein said composition comprises at least one excipient that may further aid in enhancing the targeting and/or delivery of said composition and/or said oligonucleotide to a tissue and/or cell and/or into a tissue and/or cell.
 18. The method of claim 1, wherein said oligonucleotide preferentially hybridizes to a disease-associated or disease-causing transcript and leaves a function of a normal transcript relatively unaffected.
 19. The method of claim 1, wherein said subject has DRPLA, SBMA, or SCA type 1, 2, 3, 6, 7, 12 or
 17. 20. The method of claim 19, wherein said SCA type 1 is caused by CAG repeat expansions in the transcripts of ATXN1 (SEQ ID NO:81), said SCA type 2 is caused by CAG repeat expansions in the transcripts of ATXN2 (SEQ ID NO: 82), said SCA type 3 is caused by CAG repeat expansions in the transcripts of ATXN3 (SEQ ID NO: 83), said SCA type 6 is caused by CAG repeat expansions in the transcripts of CACNA1A (SEQ ID NO:84), said SCA type 7 is caused by CAG repeat expansions in the transcripts of ATXN7 (SEQ ID NO: 85), said SCA type 12 is caused by CAG repeat expansions in the transcripts of PPP2R2B (SEQ ID NO: 86), and said SCA type 17 is caused by CAG repeat expansions in the transcripts of TBP (SEQ ID NO: 87) genes.
 21. The method of claim 1, wherein said SBMA is caused by CAG repeat expansions in the transcripts of AR (SEQ ID NO: 88), and said DRPLA is caused by CAG repeat expansions in the transcripts of ATN1 (SEQ ID NO: 89) genes.
 22. The method of claim 1, wherein the antisense oligonucleotide is provided by intrathecal administration.
 23. The method of claim 4, wherein said oligonucleotide consists of a repetitive nucleotide unit (XYG)7, wherein each X is 5-methylcytosine and each Y is a uracil (SEQ ID NO: 2).
 24. A method for treating, delaying, ameliorating and/or preventing a spinocerebellar ataxia (SCA) type 1 or type 3 in a subject, comprising providing to the subject an antisense oligonucleotide which consists of 2′-O-methyl RNA nucleotide residues and has a backbone wherein all phosphate moieties are replaced by a phosphorothioate moiety, and which comprises the base sequence of SEQ ID NO:
 90. 25. The method of claim 23, wherein the antisense oligonucleotide is provided by intrathecal administration. 